Animal feed additives

ABSTRACT

The present invention relates to methods of releasing galactose from legumes using polypeptides having alpha-galactosidase activity. The invention also relates to polypeptides having alpha-galactosidase activity, polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of 5 producing the polypeptides. The invention also relates to compositions comprising the polypeptides of the invention and the use of the polypeptides in animal feed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofinternational application no. PCT/EP2017/062652 filed May 24, 2017 whichclaims priority or the benefit under 35 U.S.C. 119 of Europeanapplication no. 16170961.3 filed May 24, 2018, the contents of which arefully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to methods of releasing galactose fromlegumes using polypeptides having alpha-galactosidase activity. Theinvention also relates to polypeptides having alpha-galactosidaseactivity, polynucleotides encoding the polypeptides, nucleic acidconstructs, vectors, and host cells comprising the polynucleotides aswell as methods of producing the polypeptides. The invention alsorelates to compositions comprising the polypeptides of the invention andthe use of the polypeptides in animal feed.

Description of the Related Art

Soybean is a species of legume native to East Asia and is the secondbiggest animal feed crop globally and the biggest feed protein source.Soy bean can be manufactured (defatted) to produce soybean meal (SBM),and SBM is a significant and cheap source of protein for animal feeds.Other common types of legume are chickpea, lupin, lentil, peanut, beansor peas which can also be processed and used as animal feed.

Alpha-galactosidase is a glycoside hydrolase enzyme that hydrolyses theterminal alpha-galactosyl moieties from glycolipids and glycoproteinsthat is present in, e.g. legumes, vegetables, grains, cereals and thelike. Alpha-galactosidases are produced by various microorganisms,plants and animals, but monogastric animals are deficient in intestinalalpha-galactosidase production and consequently are incapable ofdecomposing ingested alpha-galactosidases by themselves. Instead,ingested alpha-galactosidases are decomposed by microorganisms presentin the large intestines, where this microbial fermentation may causeflatulence and possibly wet litter.

Animal feed supplemented with alpha-galactosidases can help the animaldigest the food thereby obtaining a higher release of galactose andsucrose which will promote animal growth (performance). However, thealpha-galactoside needs to survive passage through the stomach, i.e. lowpH and proteases such as pepsin, before it can get to work in theintestine. Animal feed, especially for poultry, is normally pelletedbefore being fed and the enzyme also needs to survive the pelletingconditions. Thus the object of this invention is to provide acid stablealpha-galactosidases which are highly effective at releasing galactoseand sucrose from legumes.

SUMMARY OF THE INVENTION

The present invention relates to an animal feed additive, granules andliquid formulations comprising one or more GH36 polypeptides havingalpha-galactosidase activity selected from the group consisting of:

-   -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        3;    -   (b) a polypeptide encoded by a polynucleotide that hybridizes        under high stringency conditions, or very high stringency        conditions with        -   (i) the mature polypeptide coding sequence of SEQ ID NO: 1,        -   (ii) the cDNA sequence thereof, or        -   (iii) the full-length complementary strand of (i) or (ii);    -   (c) a polypeptide encoded by a polynucleotide having at least        80%, e.g., at least 85%, at least 86%, at least 87%, at least        88%, at least 89%, at least 90%, at least 91%, at least 92%, at        least 93%, at least 94%, at least 95%, at least 96%, at least        97%, at least 98%, at least 99%, or 100% sequence identity to        the mature polypeptide coding sequence of SEQ ID NO: 1;    -   (d) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, wherein the variant has        alpha-galactosidase activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in up to 50 positions;    -   (e) a polypeptide comprising the polypeptide of (a), (b), (c)        or (d) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;    -   (f) a polypeptide comprising the polypeptide of (a), (b), (c)        or (d) and a N-terminal and/or C-terminal extension of up to 10        amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;        and    -   (g) a fragment of the polypeptide of (a), (b), (c) or (d) having        alpha-galactosidase activity and having at least 90% of the        length of the mature polypeptide.

The invention further relates to isolated polypeptides havingalpha-galactosidase activity as defined in the claims; compositions,such as animal feed, comprising the polypeptide of the invention anduses thereof; methods of releasing galactose from plant based material;methods of improving the performance of an animal; methods for improvingthe nutritional value of an animal feed; polynucleotides encoding thepolypeptides of the present invention; nucleic acid constructs;expression vectors; recombinant host cells comprising thepolynucleotides; methods of producing the polypeptides and uses thereof.

OVERVIEW OF SEQUENCE LISTING

SEQ ID NO: 1 is the cDNA sequence of GH36 alpha-galactosidase asisolated from Penicillium pseudopulvillorum.

SEQ ID NO: 2 is the amino acid sequence as deduced from SEQ ID NO: 2.

SEQ ID NO: 3 is the amino acid sequence of the mature GH36alpha-galactosidase from Penicillium pseudopulvillorum.

SEQ ID NO: 4 is the corrected amino acid sequence of thealpha-galactosidase as disclosed in WO1994/23022.

DEFINITIONS

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-galactosidase: The term “alpha-galactosidase”, also calledα-D-galactoside galactohydrolase (E.C. 3.2.1.22), means an enzyme thatcatalyses the hydrolysis of terminal, non-reducing α-D-galactoseresidues in α-D-galactosides, such as galactose oligosaccharides,galactomannans and galactolipids. Alpha-galactosidase activity can bedetermined using 4-nitrophenyl α-D-galactopyranoside (available fromMegazyme International, Bray, Co. Wicklow, Ireland) as substrate in 100mM MES (Sigma) buffer pH 7.0±0.05 at room temperature. The enzyme isdiluted in 2-fold dilutions and then the 4-nitrophenylα-D-galactopyranoside substrate is dissolved in the solution containingthe enzyme. The alpha-galactosidase activity is followed directly in thebuffer by measuring the absorbance of released pNP at 405 nm as functionof time. A detailed assay can be found in the alpha-galactosidase assayas described herein.

In one aspect, the polypeptides of the present invention have at least40%, such as at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or at least 100% ofthe alpha-galactosidase activity of the polypeptide of SEQ ID NO: 3.

In one aspect, the polypeptide has improved gastric stability comparedto control (wherein control is defined as SEQ ID NO: 4). In anembodiment, the gastric stability is measured as the half-life at 40° C.pH3 with 0.1 mg/ml pepsin. In an embodiment, gastric stability ismeasured as described in example 4 herein. In a further embodiment, thepolypeptide has a half-life of at least 90 minutes, such as 2 hours, 3hours or 4 hours.

In one aspect, the polypeptide has increased alpha-galactosidaseactivity compared to control (wherein control is defined as SEQ ID NO:4). In one embodiment, activity is measured using 4-nitrophenylα-D-galactopyranoside as substrate. In one embodiment, activity ismeasured using 4-nitrophenyl α-D-galactopyranoside as substrate (1 mg/mlin 100 mM MES buffer pH 7.0±0.05, prepared immediately before use) assubstrate by measuring the absorbance of released pNP in the buffer at405 nm for 5 minutes as function of time at room temperature (typically23° C.). In an embodiment, activity is measured as described in example3 herein. In an embodiment, the polypeptide has an activity of at least100000 (mOD/min)/(mg/ml), such as at least 150000, at least 200000, atleast 250000, or at least 300000 (mOD/min)/(mg/ml) using 4-nitrophenylα-D-galactopyranoside as substrate.

Animal: The term “animal” refers to all animals except humans. Examplesof animals are non-ruminants, and ruminants. Ruminant animals include,for example, animals such as sheep, goats, cattle, e.g. beef cattle,cows, and young calves, deer, yank, camel, llama and kangaroo.Non-ruminant animals include mono-gastric animals, e.g. pigs or swine(including, but not limited to, piglets, growing pigs, and sows);poultry such as turkeys, ducks and chicken (including but not limited tobroiler chicks, layers); horses (including but not limited to hotbloods,coldbloods and warm bloods), young calves; fish (including but notlimited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream,bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia,cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper,guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra,mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach,salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead,snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench,terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish);and crustaceans (including but not limited to shrimps and prawns).

Animal feed: The term “animal feed” refers to any compound, preparation,or mixture suitable for, or intended for intake by an animal. Animalfeed for a mono-gastric animal typically comprises concentrates as wellas vitamins, minerals, enzymes, direct fed microbial, amino acids and/orother feed ingredients (such as in a premix) whereas animal feed forruminants generally comprises forage (including roughage and silage) andmay further comprise concentrates as well as vitamins, minerals, enzymesdirect fed microbial, amino acid and/or other feed ingredients (such asin a premix).

Body Weight Gain: The term “body weight gain” means an increase in liveweight of an animal during a given period of time e.g. the increase inweight from day 1 to day 21.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Feed Conversion Ratio: The term “feed conversion ratio” the amount offeed fed to an animal to increase the weight of the animal by aspecified amount. An improved feed conversion ratio means a lower feedconversion ratio. By “lower feed conversion ratio” or “improved feedconversion ratio” it is meant that the use of a feed additivecomposition in feed results in a lower amount of feed being required tobe fed to an animal to increase the weight of the animal by a specifiedamount compared to the amount of feed required to increase the weight ofthe animal by the same amount when the feed does not comprise said feedadditive composition.

Feed efficiency: The term “feed efficiency” means the amount of weightgain per unit of feed when the animal is fed ad-libitum or a specifiedamount of food during a period of time. By “increased feed efficiency”it is meant that the use of a feed additive composition according thepresent invention in feed results in an increased weight gain per unitof feed intake compared with an animal fed without said feed additivecomposition being present.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide or domain; wherein the fragment hasalpha-galactosidase activity.

In one aspect, the fragment comprises at least 90% of the length of themature polypeptide, such as at least 648 amino acids of SEQ ID NO: 2 orat least 648 amino acids of SEQ ID NO: 3. In another aspect, thefragment comprises at least 92% of the length of the mature polypeptide,such as at least 663 amino acids of SEQ ID NO: 2 or at least 663 aminoacids of SEQ ID NO: 3. In another aspect, the fragment comprises atleast 94% of the length of the mature polypeptide, such as at least 677amino acids of SEQ ID NO: 2 or at least 677 amino acids of SEQ ID NO: 3.

In another aspect, the fragment comprises at least 96% of the length ofthe mature polypeptide, such as at least 692 amino acids of SEQ ID NO: 2or at least 692 amino acids of SEQ ID NO: 3. In another aspect, thefragment comprises at least 98% of the length of the mature polypeptide,such as at least 706 amino acids of SEQ ID NO: 2 or at least 706 aminoacids of SEQ ID NO: 3. In another aspect, the fragment comprises atleast 99% of the length of the mature polypeptide, such as at least 713amino acids of SEQ ID NO: 2 or at least 713 amino acids of SEQ ID NO: 3.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 721 of SEQ ID NO: 2. In another aspect,the mature polypeptide is amino acids 1 to 721 of SEQ ID NO: 3.

It is known in the art that a host cell may produce a mixture of two ofmore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide. Itis also known in the art that different host cells process polypeptidesdifferently, and thus, one host cell expressing a polynucleotide mayproduce a different mature polypeptide (e.g., having a differentC-terminal and/or N-terminal amino acid) as compared to another hostcell expressing the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving alpha-galactosidase activity. In one aspect, the maturepolypeptide coding sequence is the joined sequence of nucleotides 1 to82, nucleotides 143 to 746 and nucleotides 822 to 2361 of SEQ ID NO: 1.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Nutrient Digestibility: The term “nutrient digestibility” means thefraction of a nutrient that disappears from the gastro-intestinal tractor a specified segment of the gastro-intestinal tract, e.g. the smallintestine. Nutrient digestibility may be measured as the differencebetween what is administered to the subject and what. comes out in thefaeces of the subject, or between what is administered to the subjectand what remains in the digesta on a specified segment of the gastrointestinal tract, e.g. the ileum.

Nutrient digestibility as used herein may be measured by the differencebetween the intake of a nutrient and the excreted nutrient by means ofthe total collection of excreta during a period of time; or with the useof an inert marker that is not absorbed by the animal, and allows theresearcher calculating the amount of nutrient that disappeared in theentire gastro-intestinal tract or a segment of the gastro-intestinaltract. Such an inert marker may be titanium dioxide, chromic oxide oracid insoluble ash. Digestibility may be expressed as a percentage ofthe nutrient in the feed, or as mass units of digestible nutrient permass units of nutrient in the feed. Nutrient digestibility as usedherein encompasses starch digestibility, fat digestibility, proteindigestibility, and amino acid digestibility.

Energy digestibility as used herein means the gross energy of the feedconsumed minus the gross energy of the faeces or the gross energy of thefeed consumed minus the gross energy of the remaining digesta on aspecified segment of the gastro-intestinal tract of the animal, e.g. theileum. Metabolizable energy as used herein refers to apparentmetabolizable energy and means the gross energy of the feed consumedminus the gross energy contained in the faeces, urine, and gaseousproducts of digestion. Energy digestibility and metabolizable energy maybe measured as the difference between the intake of gross energy and thegross energy excreted in the faeces or the digesta present in specifiedsegment of the gastro-intestinal tract using the same methods to measurethe digestibility of nutrients, with appropriate corrections fornitrogen excretion to calculate metabolizable energy of feed.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Release x g galactose per kg soybean meal: The term “release x ggalactose per kg soybean meal” means the amount of galactose in gramswhich is released into the supernatant after soybean meal has beenincubation with an enzyme. For the purpose of the present invention, therelease of galactose per kg soybean meal may be determined whenperformed under the reaction conditions 20 mg polypeptide per kg soybeanmeal in 10% w/v 0.1M citric acid-phosphate buffer pH 6.5 incubating at40° C. for 2 hours as described in the galactose assay herein.

In a more detailed embodiment, a 10 w/v % slurry of soybean meal isprepared from soybean meal milled to a 0.5 mm particle size and 0.1 Mcitric acid-phosphate buffer, pH 6.5±0.05. The incubation vessels withthe 10 w/v % slurry of soybean meal is heated to a stable temperature of40±2° C. while stirring. When a stable temperature had been achieved,the six D-(+)-galactose standards are added to the incubation vessels toin-vessel concentrations of 5, 2.5, 1.25, 0.625, 0.313 and 0.157 mggalactose per mL incubation volume. Each standard is incubated induplicates. The diluted enzymes are then added to their respectiveincubation vessels in the volumes required to reach their desiredconcentrations (in mg EP/kg soybean meal). Each enzyme treatment isincubated in triplicates. Additionally, two times three incubationvessels are included without standards or enzyme treatments as blanktreatments to obtain the baseline galactose concentration in the soybeanmeal slurry. The incubation vessels are incubated at 40±2° C., whilestirring for 2 hours. After incubation the vessels are centrifuged at1500 g at 5° C. for 15 minutes. The supernatants are then analyzed in anassay based on the Raffinose/Galactose kit from Megazyme (product nameK-RAFGA) and the concentration of galactose is then calculated asdescribed in the galactose assay herein.

Plant based material: The term “plant based material” means that theplant based material is from the taxonomic subclass rosids. In oneaspect, the plant based material is from the taxonomic order Fabales,such as the family Fabaceae, preferably the subfamilies Caesalpinioideaeor Mimosoideae or Papilionoideae, or more preferably from the tribesPhaseoleae, Cicereae, Genisteae, Fabeae, Dalbergieae or Phaseoleae. Inone aspect, the plant based material is from the taxonomic orderBrassicales, such as the family Brassicaceae, preferably the tribeBrassiceae, more preferably the family Brassica.

In particular embodiments, the plant based material is soybean, wildsoybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean,lima bean, French bean, Broad bean (fava bean), chickpea, lentil,peanut, Spanish peanut, canola, rapeseed (oilseed rape) or pea or in aprocessed form such as soybean meal, full fat soy bean meal, soy proteinconcentrate (SPC), fermented soybean meal (FSBM) or any combinationthereof. In a preferred embodiment, the plant based material is soybeanor soybean meal.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. Version 6.1.0 was used. The optional parameters used are gap openpenalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSSversion of BLOSUM62) substitution matrix. The output of Needle labelled“longest identity” (obtained using the −nobrief option) is used as thepercent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. Version 6.1.0 was used. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4)substitution matrix. The output of Needle labelled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Stringency conditions: The different stringency conditions are definedas follows.

The term “very low stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 25% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2.0×SSC, 0.2% SDS at 60° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2.0×SSC, 0.2% SDS at 65° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 1.0×SSC, 0.2% SDS at 65° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 1.0×SSC, 0.2% SDS at 70° C.

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 0.5×SSC, 0.2% SDS at 70° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 0.5×SSC, 0.2% SDS at 75° C.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having alpha-galactosidase activity.

Substantially pure polypeptide: The term “substantially purepolypeptide” means a preparation that contains at most 10%, at most 8%,at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%,and at most 0.5% by weight of other polypeptide material with which itis natively or recombinantly associated. Preferably, the polypeptide isat least 92% pure, e.g., at least 94% pure, at least 95% pure, at least96% pure, at least 97% pure, at least 98% pure, at least 99%, at least99.5% pure, and 100% pure by weight of the total polypeptide materialpresent in the preparation. The polypeptides of the present inventionare preferably in a substantially pure form. This can be accomplished,for example, by preparing the polypeptide by well-known recombinantmethods or by classical purification methods.

Variant: The term “variant” means a polypeptide havingalpha-galactosidase activity comprising an alteration, i.e., asubstitution, insertion, and/or deletion of one or more (several) aminoacid residues at one or more (several) positions. A substitution means areplacement of an amino acid occupying a position with a different aminoacid; a deletion means removal of an amino acid occupying a position;and an insertion means adding 1-3 amino acids adjacent to an amino acidoccupying a position.

In one aspect, the variants of the present invention have at least 40%,such as at least 50%, at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95% or at least 100% of thealpha-galactosidase activity of the polypeptide of SEQ ID NO: 3 whenusing 4-nitrophenyl α-D-galactopyranoside as substrate as describedherein.

In one aspect, the variant has improved gastric stability compared tocontrol (wherein control is defined as SEQ ID NO: 4). In an embodiment,the gastric stability is measured as the half-life at 40° C. pH3 with0.1 mg/ml pepsin. In an embodiment, gastric stability is measured asdescribed in example 4 herein. In a further embodiment, the variant hasa half-life of at least 90 minutes, such as 2 hours, 3 hours or 4 hours.

In one aspect, the variant has increased alpha-galactosidase activitycompared to control (wherein control is defined as SEQ ID NO: 4). In oneembodiment, activity is measured using 4-nitrophenylα-D-galactopyranoside as substrate. In one embodiment, activity ismeasured using 4-nitrophenyl α-D-galactopyranoside as substrate (1 mg/mlin 100 mM MES buffer pH 7.0±0.05, prepared immediately before use) assubstrate by measuring the absorbance of released pNP in the buffer at405 nm for 5 minutes as function of time at room temperature (typically23° C.). In an embodiment, activity is measured as described in example3 herein. In an embodiment, the variant has an activity of at least100000 (mOD/min)/(mg/ml), such as at least 150000, at least 200000, atleast 250000, or at least 300000 (mOD/min)/(mg/ml) using 4-nitrophenylα-D-galactopyranoside as substrate.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that certain alpha-galactosidases fromglycoside hydrolase family 36 (herein referred to as GH36) aresurprisingly good at degrading the raffinose family of oligosaccharides(RFOs), such as the trisaccharide raffinose, the tetrasaccharidestachyose, and the pentasaccharide verbascose. These RFOs are typicallyfound in plants from the taxonomic subclass rosids, specifically thesubfamily Papilionoideae, such as in soy beans, chickpea, beans, lupin,lentil, peanut and peas or the tribe Brassiceae, such as in canola orrapeseed.

Efficient alpha-galactosidases are required to effectively degradeRFO's. However, the alpha-galactoside also needs to survive passagethrough the stomach of the animal where there is a low pH and proteasessuch as pepsin, before it can get to work in the intestine. If thealpha-galactosidase is degraded or denatured whilst passing through thestomach then it will not be present to work in the intestine of theanimal.

The degradation of RFO's can be measured as the amount of galactosereleased into the supernatant when e.g. soybean meal is treated with analpha-galactosidase. Increased amounts of solubilisation will result inmore galactose being released which can be detected using e.g. theGalactose SBM Assay method as described herein. Thus the inventionrelates to an acid stable alpha-galactosidase which is also highlyefficient at releasing galactose from plant material, such as soybeanmeal.

Animal Feed and Animal Feed Additives

In a first aspect, the invention relates to animal feed additivescomprising one or more GH36 polypeptides having alpha-galactosidaseactivity, wherein the polypeptide having alpha-galactosidase activityhas at least 80%, e.g., at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 2. In one embodiment, the polypeptides differby up to 50 amino acids, e.g., between 1 and 50 amino acids, such as1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 amino acids, or 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids from the maturepolypeptide of SEQ ID NO: 2.

In a continuation of the first aspect, the invention relates to animalfeed additives comprising one or more GH36 polypeptides havingalpha-galactosidase activity, wherein the polypeptide havingalpha-galactosidase activity having at least 82%, e.g., at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 3. In one embodiment, the polypeptides differ byup to 50 amino acids, e.g., between 1 and 50 amino acids, such as 1-45,1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 amino acids, or 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids from SEQ ID NO: 3.

In one embodiment, the polypeptide preferably comprises or consists ofthe amino acid sequence of SEQ ID NO: 3 or an allelic variant thereof;comprises the amino acid sequence of SEQ ID NO: 3 and a N-terminaland/or C-terminal His-tag and/or HQ-tag; or is a fragment thereof havingalpha-galactosidase activity and having at least 90% such as at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% of the length of themature polypeptide. In another embodiment, the polypeptide comprises orconsists of the mature polypeptide of SEQ ID NO: 3. In anotherembodiment, the polypeptide comprises or consists of amino acids 1 to721 of SEQ ID NO: 2. In another embodiment, the polypeptide comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In anotherembodiment, the polypeptide comprises or consists of amino acids 1 to721 of SEQ ID NO: 3. In an embodiment, the polypeptide has beenisolated.

In a continuation of the first aspect, the invention relates to animalfeed additives comprising one or more GH36 polypeptides havingalpha-galactosidase activity, wherein the polypeptide havingalpha-galactosidase activity encoded by a polynucleotide that hybridizesunder high or very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii) (Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, New York). In an embodiment, the polypeptide has beenisolated.

In a continuation of the first aspect, the invention relates to animalfeed additives comprising one or more GH36 polypeptides havingalpha-galactosidase activity, wherein the polypeptide havingalpha-galactosidase activity encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 80%, e.g., at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%. In a further embodiment, thepolypeptide has been isolated.

In one embodiment of the first aspect of the invention, the polypeptidehas improved gastric stability compared to control (wherein control isdefined as SEQ ID NO: 4). In an embodiment, the gastric stability ismeasured as the half-life at 40° C. pH3 with 0.1 mg/ml pepsin. In anembodiment, gastric stability is measured as described in example 4herein. In a further embodiment, the polypeptide has a half-life of atleast 90 minutes, such as 2 hours, 3 hours or 4 hours.

In one embodiment of the first aspect of the invention, the polypeptidehas increased alpha-galactosidase activity compared to control (whereincontrol is defined as SEQ ID NO: 4). In one embodiment, activity ismeasured using 4-nitrophenyl α-D-galactopyranoside as substrate.

In one embodiment, activity is measured using 4-nitrophenylα-D-galactopyranoside as substrate (1 mg/ml in 100 mM MES buffer pH7.0±0.05, prepared immediately before use) as substrate by measuring theabsorbance of released pNP in the buffer at 405 nm for 5 minutes asfunction of time at room temperature (typically 23° C.). In anembodiment, activity is measured as described in example 3 herein. In anembodiment, the polypeptide has an activity of at least 100000(mOD/min)/(mg/ml), such as at least 150000, at least 200000, at least250000, or at least 300000 (mOD/min)/(mg/ml) using 4-nitrophenylα-D-galactopyranoside as substrate. In an embodiment, the polypeptidehas at least 40%, such as at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 100% of the alpha-galactosidase activity of the polypeptide of SEQID NO: 3 (as determined using 4-nitrophenyl α-D-galactopyranoside assubstrate).

In a continuation of the first aspect, the invention relates to animalfeed additives comprising variants of SEQ ID NO: 3, wherein the variantof SEQ ID NO: 3 has alpha-galactosidase activity and comprises one ormore amino acid substitutions, and/or one or more amino acid deletions,and/or one or more amino acid insertions or any combination thereof atone or more (e.g., several) positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 3 is not more than 50, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In another embodiment, thenumber of positions comprising one or more amino acid substitutions,and/or one or more amino acid deletions, and/or one or more amino acidinsertions or any combination thereof in SEQ ID NO: 3 is between 1 and45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 positions.In an embodiment, the number of positions comprising one or more aminoacid substitutions, and/or one or more amino acid deletions, and/or oneor more amino acid insertions or any combination thereof in SEQ ID NO: 3is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In anotherembodiment, the number of substitutions and/or deletions and/orinsertions in SEQ ID NO: 3 is not more than 10, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9 or 10. In a further embodiment, the number of substitutions inSEQ ID NO: 3 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.In a further embodiment, the number of conservative substitutions in SEQID NO: 3 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly. Other examples of conservativesubstitutions are G to A; AtoG, S; V to I, L, A, T, S; I to V, L, M; Lto I, M, V; M to L, I, V; P to A, S, N; F to Y, W, H; Y to F, W, H; W toY, F, H; R to K, E, D; K to R, E, D; H to Q, N, S; D to N, E, K, R, Q; Eto Q, D, K, R, N; S to T, A; T to S, V, A; C to S, T, A; N to D, Q, H,S; Q to E, N, H, K, R.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for alpha-galactosidase activity to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labelling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

In one embodiment of the first aspect of the invention, the variant hasimproved gastric stability compared to control (wherein control isdefined as SEQ ID NO: 4). In an embodiment, the gastric stability ismeasured as the half-life at 40° C. pH3 with 0.1 mg/ml pepsin. In anembodiment, gastric stability is measured as described in example 4herein. In a further embodiment, the variant has a half-life of at least90 minutes, such as 2 hours, 3 hours or 4 hours.

In one embodiment of the first aspect of the invention, the variant hasincreased alpha-galactosidase activity compared to control (whereincontrol is defined as SEQ ID NO: 4). In one embodiment, activity ismeasured using 4-nitrophenyl α-D-galactopyranoside as substrate. In oneembodiment, activity is measured using 4-nitrophenylα-D-galactopyranoside as substrate (1 mg/ml in 100 mM MES buffer pH7.0±0.05, prepared immediately before use) as substrate by measuring theabsorbance of released pNP in the buffer at 405 nm for 5 minutes asfunction of time at room temperature (typically 23° C.). In anembodiment, activity is measured as described in example 3 herein. In anembodiment, the variant has an activity of at least 100000(mOD/min)/(mg/ml), such as at least 150000, at least 200000, at least250000, or at least 300000 (mOD/min)/(mg/ml) using 4-nitrophenylα-D-galactopyranoside as substrate. In an embodiment, the variant has atleast 40%, such as at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or at least100% of the alpha-galactosidase activity of the variant of SEQ ID NO: 3(as determined using 4-nitrophenyl α-D-galactopyranoside as substrate).

In an embodiment, the plant based material is from the taxonomicsubclass rosids. In one aspect, the plant based material is from thetaxonomic order Fabales, such as the family Fabaceae, preferably thesubfamilies Caesalpinioideae or Mimosoideae or Papilionoideae, or morepreferably from the tribes Phaseoleae, Cicereae, Genisteae, Fabeae,Dalbergieae or Phaseoleae. In one aspect, the plant based material isfrom the taxonomic order Brassicales, such as the family Brassicaceae,preferably the tribe Brassiceae, more preferably the family Brassica.

In particular embodiments, the plant based material is soybean, wildsoybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean,lima bean, French bean, Broad bean (fava bean), chickpea, lentil,peanut, Spanish peanut, canola, rapeseed (oilseed rape) or pea or in aprocessed form such as soybean meal, full fat soy bean meal, soy proteinconcentrate (SPC), fermented soybean meal (FSBM) or any combinationthereof. In a preferred embodiment, the plant based material is soybeanor soybean meal.

Polypeptides Having Alpha-Galactosidase Activity

In a second aspect, the invention relates to polypeptides havingalpha-galactosidase activity having at least 96.5%, e.g., at least 97%,at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least99.5%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:2. In one embodiment, the polypeptides differ by up to 27 amino acids,e.g., between 1 and 27 amino acids, such as 1-25, 1-20, 1-15, 1-10 or1-5 amino acids, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 amino acids from themature polypeptide of SEQ ID NO: 2.

In an embodiment, the polypeptide releases at least 14 g, such as atleast 14.5 g, at least 15 g, at least 16 g, at least 17 g, at least 18g, at least 19 g, at least 20 g or at least 22 g galactose per kgsoybean meal when performed under the reaction conditions 20 mgpolypeptide per kg soybean meal in 10% w/v 0.1M citric acid-phosphatebuffer pH 6.5 incubating at 4⁰° C. for 2 hours.

In a continuation of the second aspect, the invention relates topolypeptides having alpha-galactosidase activity having at least 96.5%,e.g., at least 97%, at least 97.5%, at least 98%, at least 98.5%, atleast 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO: 3. Inone embodiment, the polypeptides differ by up to 27 amino acids, e.g.,between 1 and 27 amino acids, such as 1-25, 1-20, 1-15, 1-10 or 1-5amino acids, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 amino acids from the maturepolypeptide of SEQ ID NO: 3.

In one embodiment, the polypeptide preferably comprises or consists ofthe amino acid sequence of SEQ ID NO: 3 or an allelic variant thereof;comprises the amino acid sequence of SEQ ID NO: 3 and a N-terminaland/or C-terminal His-tag and/or HQ-tag; or is a fragment thereof havingalpha-galactosidase activity and having at least 90% such as at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% of the length of themature polypeptide. In another embodiment, the polypeptide comprises orconsists of the mature polypeptide of SEQ ID NO: 3. In anotherembodiment, the polypeptide comprises or consists of amino acids 1 to721 of SEQ ID NO: 2. In another embodiment, the polypeptide comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In anotherembodiment, the polypeptide comprises or consists of amino acids 1 to721 of SEQ ID NO: 3. In an embodiment, the polypeptide has beenisolated.

In a continuation of the second aspect, the invention relates to apolypeptide having alpha-galactosidase activity encoded by apolynucleotide having a sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 of at least 96.5%, e.g., at least 97%,at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least99.5%, or 100%. In a further embodiment, the polypeptide has beenisolated.

In a continuation of the second aspect, the invention relates tovariants of SEQ ID NO: 3 having alpha-galactosidase activity comprisingone or more amino acid substitutions, and/or one or more amino aciddeletions, and/or one or more amino acid insertions or any combinationthereof at one or more (e.g., several) positions. In an embodiment, thenumber of positions comprising one or more amino acid substitutions,and/or one or more amino acid deletions, and/or one or more amino acidinsertions or any combination thereof in SEQ ID NO: 3 is not more than27, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26 or 27. In another embodiment, the numberof positions comprising one or more amino acid substitutions, and/or oneor more amino acid deletions, and/or one or more amino acid insertionsor any combination thereof in SEQ ID NO: 3 is between 1 and 25, such as1-20, 1-15, 1-10 or 1-5 positions. In an embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 3 is not more than 10, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number ofsubstitutions and/or deletions and/or insertions in SEQ ID NO: 3 is notmore than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a furtherembodiment, the number of substitutions in SEQ ID NO: 3 is not more than10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a further embodiment, thenumber of conservative substitutions in SEQ ID NO: 3 is not more than10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Examples of amino acidchanges, conservative substitutions and fusion peptides are described inthe first aspect herein.

Granules Comprising Polypeptides Having Alpha-Galactosidase Activity

In a third aspect, the invention relates to a granule comprising a GH36polypeptide having having alpha-galactosidase activity, wherein thepolypeptide is selected from the group consisting of:

-   -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        3;    -   (b) a variant of SEQ ID NO: 3, wherein the variant has        alpha-galactosidase activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,        48, 49 or 50 positions;    -   (c) a polypeptide comprising the polypeptide of (a) or (b) and a        N-terminal and/or C-terminal His-tag and/or HQ-tag;    -   (d) a polypeptide comprising the polypeptide of (a) or (b) and a        N-terminal and/or C-terminal extension of up to 10 amino acids,        e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and    -   (e) a fragment of the polypeptide of (a) or (b) having        alpha-galactosidase activity and having at least 90% of the        length of the mature polypeptide.

In one embodiment, the granule comprises a core particle and one or morecoatings. In a preferred embodiment, the coating comprises salt and/orwax and/or flour. Preferred formulations are disclosed in theformulation section below.

In one embodiment of the third aspect of the invention, the polypeptidehas improved gastric stability compared to control (wherein control isdefined as SEQ ID NO: 4). In an embodiment, the gastric stability ismeasured as the half-life at 40° C. pH3 with 0.1 mg/ml pepsin.

In an embodiment, gastric stability is measured as described in example4 herein. In a further embodiment, the polypeptide has a half-life of atleast 90 minutes, such as 2 hours, 3 hours or 4 hours.

In one embodiment of the third aspect of the invention, the polypeptidehas increased alpha-galactosidase activity compared to control (whereincontrol is defined as SEQ ID NO: 4). In one embodiment, activity ismeasured using 4-nitrophenyl α-D-galactopyranoside as substrate.

In one embodiment, activity is measured using 4-nitrophenylα-D-galactopyranoside as substrate (1 mg/ml in 100 mM MES buffer pH7.0±0.05, prepared immediately before use) as substrate by measuring theabsorbance of released pNP in the buffer at 405 nm for 5 minutes asfunction of time at room temperature (typically 23° C.). In anembodiment, activity is measured as described in example 3 herein. In anembodiment, the polypeptide has an activity of at least 100000(mOD/min)/(mg/ml), such as at least 150000, at least 200000, at least250000, or at least 300000 (mOD/min)/(mg/ml) using 4-nitrophenylα-D-galactopyranoside as substrate. In an embodiment, the polypeptidehas at least 40%, such as at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 100% of the alpha-galactosidase activity of the polypeptide of SEQID NO: 3 (as determined using 4-nitrophenyl α-D-galactopyranoside assubstrate).

In one embodiment of the third aspect of the invention, the compositioncomprises at least 0.01 mg of polypeptide (enzyme protein) per kilogramof composition, such as at least 0.02 mg, 0.05 mg, 0.10 mg, 0.2 mg, 0.5mg, 1.0 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1.0g, 2.5 g, 5 g, 7.5 g, 10 g, 25 g, 50 g, 75 g or 100 g per kilogram ofcomposition. In one embodiment, the composition comprises at most 250 gof polypeptide per kilogram of composition, such as at most 150 g, 100g, 50 g, 40 g, 30 g, 20 g, 10 g, 7.5 g, 5 g, 2.5 g, 1.0 g, 750 mg, 500mg, 250 mg, 100 mg, 50 mg, 25 mg, 10 mg, 5 mg, 2.5 mg or 1 mg perkilogram of composition. In one embodiment, the composition comprisesbetween 0.01 mg and 250 g of polypeptide (enzyme protein) per kilogramof composition, such as between 0.02 mg, 0.05 mg, 0.10 mg, 0.2 mg, 0.5mg, 1.0 mg, 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1.0g, 2.5 g, 5 g, 7.5 g, 10 g, 25 g, 50 g, 75 g or 100 g per kilogram ofcomposition and 150 g, 100 g, 50 g, 40 g, 30 g, 20 g, 10 g, 7.5 g, 5 g,2.5 g, 1.0 g, 750 mg, 500 mg, 250 mg, 100 mg, 50 mg, 25 mg, 10 mg, 5 mg,2.5 mg or 1 mg per kilogram of composition, or any combination thereof.

In one embodiment of the third aspect of the invention, the granulecomprises one or more formulating agents (such as those describedherein), preferably a formulating agent selected from the listconsisting of glycerol, ethylene glycol, 1, 2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate,sodium sulfate, potassium sulfate, magnesium sulfate, sodiumthiosulfate, calcium carbonate, sodium citrate, dextrin, glucose,sucrose, sorbitol, lactose, starch, kaolin, maltodextrin, cyclodextrin,wheat, PVA, acetate, phosphate and cellulose, preferably selected fromthe list consisting of 1, 2-propylene glycol, 1, 3-propylene glycol,sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin andcalcium carbonate.

In one embodiment of the third aspect of the invention, the granulecomprises a core particle and one or more coatings. In a preferredembodiment, the coating comprises salt and/or wax and/or flour.Preferred formulations are disclosed in the formulation section below.

In one embodiment of the third aspect of the invention, the granulecomprises one or more additional enzymes. The one or more additionalenzymes is preferably selected from the group consisting of acetylxylanesterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase,arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase,galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase,beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase,beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipaseA2, phospholipase D, protease, pullulanase, pectinesterase,triacylglycerol lipase, xylanase, beta-xylosidase or any combinationthereof.

In one embodiment of the third aspect of the invention, the granulecomprises one or more probiotics. The one or more probiotics ispreferably selected from the group consisting of Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus,Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacteriumanimalis, Bifidobacterium sp., Carnobacterium sp., Clostridiumbutyricum, Clostridium sp., Enterococcus faecium, Enterococcus sp.,Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus farciminus,Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillussalivarius, Lactococcus lactis, Lactococcus sp., Leuconostoc sp.,Megasphaera elsdenii, Megasphaera sp., Pediococsus acidilactici,Pediococcus sp., Propionibacterium thoenii, Propionibacterium sp. andStreptococcus sp. or any combination thereof.

Liquid formulations comprising polypeptides Having Alpha-galactosidaseActivity

In a fourth aspect, the invention relates to a liquid formulationcomprising one or more GH36 polypeptides having alpha-galactosidaseactivity, wherein the liquid formulation comprises:

-   -   (A) 0.001% to 25% w/w of polypeptide having alpha-galactosidase        activity wherein the polypeptide having alpha-galactosidase        activity is selected from the group consisting of:        -   (a) a polypeptide having at least 80%, e.g., at least 85%,            at least 86%, at least 87%, at least 88%, at least 89%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 96%, at least 97%, at            least 98%, at least 99%, or 100% sequence identity to the            polypeptide of SEQ ID NO: 3;        -   (b) a variant of SEQ ID NO: 3, wherein the variant has            alpha-galactosidase activity and comprises one or more amino            acid substitutions, and/or one or more amino acid deletions,            and/or one or more amino acid insertions or any combination            thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,            15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,            30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,            45, 46, 47, 48, 49 or 50 positions;        -   (c) a polypeptide comprising the polypeptide of (a) or (b)            and a N-terminal and/or C-terminal His-tag and/or HQ-tag;        -   (d) a polypeptide comprising the polypeptide of (a) or (b)            and a N-terminal and/or C-terminal extension of up to 10            amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino            acids; and        -   (e) a fragment of the polypeptide of (a) or (b) having            alpha-galactosidase activity and having at least 90% of the            length of the mature polypeptide; and    -   (B) water.

In one embodiment, the liquid formulation comprises 20%-80% polyol. Inone embodiment, the liquid formulation comprises 0.001% to 2.0% w/wpreservative.

In one embodiment, the invention relates to a liquid formulationcomprising one or more GH36 polypeptides having alpha-galactosidaseactivity, wherein the liquid formulation comprises:

-   -   (A) 0.001% to 25% w/w of polypeptide having alpha-galactosidase        activity wherein the polypeptide having alpha-galactosidase        activity is selected from the group consisting of:        -   (a) a polypeptide having at least 80%, e.g., at least 85%,            at least 86%, at least 87%, at least 88%, at least 89%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 96%, at least 97%, at            least 98%, at least 99%, or 100% sequence identity to the            polypeptide of SEQ ID NO: 3;        -   (b) a variant of SEQ ID NO: 3, wherein the variant has            alpha-galactosidase activity and comprises one or more amino            acid substitutions, and/or one or more amino acid deletions,            and/or one or more amino acid insertions or any combination            thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,            15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,            30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,            45, 46, 47, 48, 49 or 50 positions;        -   (c) a polypeptide comprising the polypeptide of (a) or (b)            and a N-terminal and/or C-terminal His-tag and/or HQ-tag;        -   (d) a polypeptide comprising the polypeptide of (a) or (b)            and a N-terminal and/or C-terminal extension of up to 10            amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino            acids; and        -   (e) a fragment of the polypeptide of (a) or (b) having            alpha-galactosidase activity and having at least 90% of the            length of the mature polypeptide; and    -   (B) 20% to 80% w/w of polyol;    -   (C) 0.001% to 2.0% w/w preservative; and    -   (D) water.

In one embodiment of the fourth aspect of the invention, the polypeptidehas improved gastric stability compared to control (wherein control isdefined as SEQ ID NO: 4). In an embodiment, the gastric stability ismeasured as the half-life at 40° C. pH3 with 0.1 mg/ml pepsin.

In an embodiment, gastric stability is measured as described in example4 herein. In a further embodiment, the polypeptide has a half-life of atleast 90 minutes, such as 2 hours, 3 hours or 4 hours.

In one embodiment of the fourth aspect of the invention, the polypeptidehas increased alpha-galactosidase activity compared to control (whereincontrol is defined as SEQ ID NO: 4). In one embodiment, activity ismeasured using 4-nitrophenyl α-D-galactopyranoside as substrate.

In one embodiment, activity is measured using 4-nitrophenylα-D-galactopyranoside as substrate (1 mg/ml in 100 mM MES buffer pH7.0±0.05, prepared immediately before use) as substrate by measuring theabsorbance of released pNP in the buffer at 405 nm for 5 minutes asfunction of time at room temperature (typically 23° C.). In anembodiment, activity is measured as described in example 3 herein. In anembodiment, the polypeptide has an activity of at least 100000(mOD/min)/(mg/ml), such as at least 150000, at least 200000, at least250000, or at least 300000 (mOD/min)/(mg/ml) using 4-nitrophenylα-D-galactopyranoside as substrate. In an embodiment, the polypeptidehas at least 40%, such as at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 100% of the alpha-galactosidase activity of the polypeptide of SEQID NO: 3 (as determined using 4-nitrophenyl α-D-galactopyranoside assubstrate).

In one embodiment of the fourth aspect of the invention, the liquidformulation comprises one or more formulating agents (such as thosedescribed herein), preferably a formulating agent selected from the listconsisting of glycerol, ethylene glycol, 1, 2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate,sodium sulfate, potassium sulfate, magnesium sulfate, sodiumthiosulfate, calcium carbonate, sodium citrate, dextrin, glucose,sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate,preferably selected from the list consisting of 1, 2-propylene glycol,1, 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodiumthiosulfate, kaolin and calcium carbonate.

In one embodiment of the fourth aspect of the invention, the liquidformulation comprises one or more polyols, preferably a polyol selectedfrom the group consisting of glycerol, sorbitol, propylene glycol (MPG),ethylene glycol, diethylene glycol, triethylene glycol, 1, 2-propyleneglycol or 1, 3-propylene glycol, dipropylene glycol, polyethylene glycol(PEG) having an average molecular weight below about 600 andpolypropylene glycol (PPG) having an average molecular weight belowabout 600, more preferably selected from the group consisting ofglycerol, sorbitol and propylene glycol (MPG) or any combinationthereof.

In one embodiment of the fourth aspect of the invention, the liquidformulation comprises 20%-80% polyol (i.e. total amount of polyol),preferably 25%-75% polyol, more preferably 30%-70% polyol, morepreferably 35%-65% polyol or most preferably 40%-60% polyol. In oneembodiment, the liquid formulation comprises 20%-80% polyol, preferably25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65%polyol or most preferably 40%-60% polyol wherein the polyol is selectedfrom the group consisting of glycerol, sorbitol, propylene glycol (MPG),ethylene glycol, diethylene glycol, triethylene glycol, 1, 2-propyleneglycol or 1, 3-propylene glycol, dipropylene glycol, polyethylene glycol(PEG) having an average molecular weight below about 600 andpolypropylene glycol (PPG) having an average molecular weight belowabout 600. In one embodiment, the liquid formulation comprises 20%-80%polyol (i.e. total amount of polyol), preferably 25%-75% polyol, morepreferably 30%-70% polyol, more preferably 35%-65% polyol or mostpreferably 40%-60% polyol wherein the polyol is selected from the groupconsisting of glycerol, sorbitol and propylene glycol (MPG).

In one embodiment of the fourth aspect of the invention, thepreservative is selected from the group consisting of sodium sorbate,potassium sorbate, sodium benzoate and potassion benzoate or anycombination thereof. In one embodiment, the liquid formulation comprises0.02% to 1.5% w/w preservative, more preferably 0.05% to 1.0% w/wpreservative or most preferably 0.1% to 0.5% w/w preservative. In oneembodiment, the liquid formulation comprises 0.001% to 2.0% w/wpreservative (i.e. total amount of preservative), preferably 0.02% to1.5% w/w preservative, more preferably 0.05% to 1.0% w/w preservative ormost preferably 0.1% to 0.5% w/w preservative wherein the preservativeis selected from the group consisting of sodium sorbate, potassiumsorbate, sodium benzoate and potassion benzoate or any combinationthereof.

In one embodiment of the fourth aspect of the invention, the liquidformulation comprises 0.01% to 25% w/w polypeptide havingalpha-galactosidase activity, preferably 0.05% to 20% w/w polypeptidehaving alpha-galactosidase activity, more preferably 0.2% to 15% w/wpolypeptide having alpha-galactosidase activity, more preferably 0.5% to15% w/w polypeptide having alpha-galactosidase activity or mostpreferably 1.0% to 10% w/w polypeptide having alpha-galactosidaseactivity.

In one embodiment, the liquid formulation comprises one or moreadditional enzymes. The one or more additional enzymes is preferablyselected from the group consisting of acetylxylan esterase, acylglycerollipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase,cellobiohydrolases, cellulase, feruloyl esterase, galactanase,alpha-galactosidase, beta-galactosidase, beta-glucanase,beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase,beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipaseA2, phospholipase D, protease, pullulanase, pectinesterase,triacylglycerol lipase, xylanase, beta-xylosidase or any combinationthereof.

In one embodiment of the fourth aspect of the invention, the liquidformulation comprises one or more probiotics. The one or more probioticsis preferably selected from the group consisting of Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus,Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacteriumanimalis, Bifidobacterium sp., Carnobacterium sp., Clostridiumbutyricum, Clostridium sp., Enterococcus faecium, Enterococcus sp.,Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus farciminus,Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillussalivarius, Lactococcus lactis, Lactococcus sp., Leuconostoc sp.,Megasphaera elsdenii, Megasphaera sp., Pediococsus acidilactici,Pediococcus sp., Propionibacterium thoenii, Propionibacterium sp. andStreptococcus sp. or any combination thereof.

Sources of Polypeptides Having alpha-galactosidase Activity Apolypeptide having alpha-galactosidase activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

In one embodiment, the polypeptide is from a fungus of the classEurotiomycetes, such as from the order Eurotiales, or from the familyAspergillaceae, or from the genus Penicillium or from the speciesPenicillium pseudopulvillorum.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including variant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and variant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucormieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMIR1permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally, (b) recovering thepolypeptide. In one aspect, the cell is a Penicillium cell. In anotheraspect, the cell is a Penicillium pseudopulvillorum cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally, (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides alpha-galactosidase activity. Thesedetection methods include, but are not limited to, use of specificantibodies, formation of an enzyme product, or disappearance of anenzyme substrate. For example, an enzyme assay may be used to determinethe activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Production in Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.

Plant cells and specific plant cell compartments, such as chloroplasts,apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are alsoconsidered to be a plant part.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. In some embodiments, the composition is a cell-killed wholebroth containing organic acid(s), killed cells and/or cell debris, andculture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis. In some embodiments, the cell-killed whole broth orcomposition contains the spent cell culture medium, extracellularenzymes, and killed filamentous fungal cells. In some embodiments, themicrobial cells present in the cell-killed whole broth or compositioncan be permeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in the polypeptide of the invention. The term “enriched”indicates that the alpha-galactosidase activity of the composition hasbeen increased, e.g., with an enrichment factor of at least 1.1, such asat least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, atleast 3.0, at least 4.0, at least 5.0, at least 10.

In an embodiment, the composition comprises one or more polypeptides ofthe second aspect of the invention and one or more formulating agents,as described below.

The compositions may further comprise multiple enzymatic activities,such as one or more (e.g., several) enzymes selected from the groupconsisting of acetylxylan esterase, acylglycerol lipase, amylase,alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases,cellulase, feruloyl esterase, galactanase, alpha-galactosidase,beta-galactosidase, beta-glucanase, beta-glucosidase, lysophospholipase,lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase,phospholipase A1, phospholipase A2, phospholipase D, protease,pullulanase, pectinesterase, triacylglycerol lipase, xylanase,beta-xylosidase or any combination thereof.

The compositions may further comprise one or more microbes. In anembodiment, the microbe is selected from the group consisting ofBacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens,Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillusmegaterium, Bacillus coagulans, Bacillus circulans, Bifidobacteriumbifidum, Bifidobacterium animalis, Bifidobacterium sp., Carnobacteriumsp., Clostridium butyricum, Clostridium sp., Enterococcus faecium,Enterococcus sp., Lactobacillus sp., Lactobacillus acidophilus,Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillusreuteri, Lactobacillus salivarius, Lactococcus lactis, Lactococcus sp.,Leuconostoc sp., Megasphaera elsdenii, Megasphaera sp., Pediococsusacidilactici, Pediococcus sp., Propionibacterium thoenii,Propionibacterium sp. and Streptococcus sp. or any combination thereof.

Formulation

The enzyme of the invention may be formulated as a liquid or a solid.For a liquid formulation, the formulating agent may comprise a polyol(such as e.g. glycerol, ethylene glycol or propylene glycol), a salt(such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or asugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, andsorbitol). Thus in one embodiment, the composition is a liquidcomposition comprising the polypeptide of the invention and one or moreformulating agents selected from the list consisting of glycerol,ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, sodiumchloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose,and sorbitol. The liquid formulation may be sprayed onto the feed afterit has been pelleted or may be added to drinking water given to theanimals.

For a solid formulation, the formulation may be for example as agranule, spray dried powder or agglomerate (e.g. as disclosed inWO2000/70034). The formulating agent may comprise a salt (organic orinorganic zinc, sodium, potassium or calcium salts such as e.g. such ascalcium acetate, calcium benzoate, calcium carbonate, calcium chloride,calcium citrate, calcium sorbate, calcium sulfate, potassium acetate,potassium benzoate, potassium carbonate, potassium chloride, potassiumcitrate, potassium sorbate, potassium sulfate, sodium acetate, sodiumbenzoate, sodium carbonate, sodium chloride, sodium citrate, sodiumsulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride,zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugarderivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol).

In one embodiment, the composition is a solid composition, such as aspray dried composition, comprising the alpha-galactosidase of theinvention and one or more formulating agents selected from the listconsisting of sodium chloride, sodium benzoate, potassium sorbate,sodium sulfate, potassium sulfate, magnesium sulfate, sodiumthiosulfate, calcium carbonate, sodium citrate, dextrin, glucose,sucrose, sorbitol, lactose, starch and cellulose. In a preferredembodiment, the formulating agent is selected from one or more of thefollowing compounds: sodium sulfate, dextrin, cellulose, sodiumthiosulfate, magnesium sulfate and calcium carbonate.

The present invention also relates to enzyme granules/particlescomprising the alpha-galactosidase of the invention optionally combinedwith one or more additional enzymes. The granule is composed of a core,and optionally one or more coatings (outer layers) surrounding the core.

Typically the granule/particle size, measured as equivalent sphericaldiameter (volume based average particle size), of the granule is 20-2000μm, particularly 50-1500 μm, 100-1500 μm or 250-1200 μm.

The core can be prepared by granulating a blend of the ingredients,e.g., by a method comprising granulation techniques such ascrystallization, precipitation, pan-coating, fluid bed coating, fluidbed agglomeration, rotary atomization, extrusion, prilling,spheronization, size reduction methods, drum granulation, and/or highshear granulation.

Methods for preparing the core can be found in Handbook of PowderTechnology; Particle size enlargement by C. E. Capes; Volume 1; 1980;Elsevier. Preparation methods include known feed and granule formulationtechnologies, e.g.:

a) spray dried products, wherein a liquid enzyme-containing solution isatomized in a spray drying tower to form small droplets which duringtheir way down the drying tower dry to form an enzyme-containingparticulate material;

b) layered products, wherein the enzyme is coated as a layer around apre-formed inert core particle, wherein an enzyme-containing solution isatomized, typically in a fluid bed apparatus wherein the pre-formed coreparticles are fluidized, and the enzyme-containing solution adheres tothe core particles and dries up to leave a layer of dry enzyme on thesurface of the core particle. Particles of a desired size can beobtained this way if a useful core particle of the desired size can befound. This type of product is described in, e.g., WO 97/23606;

c) absorbed core particles, wherein rather than coating the enzyme as alayer around the core, the enzyme is absorbed onto and/or into thesurface of the core. Such a process is described in WO 97/39116.

d) extrusion or pelletized products, wherein an enzyme-containing pasteis pressed to pellets or under pressure is extruded through a smallopening and cut into particles which are subsequently dried. Suchparticles usually have a considerable size because of the material inwhich the extrusion opening is made (usually a plate with bore holes)sets a limit on the allowable pressure drop over the extrusion opening.Also, very high extrusion pressures when using a small opening increaseheat generation in the enzyme paste, which is harmful to the enzyme;

e) prilled products, wherein an enzyme-containing powder is suspended inmolten wax and the suspension is sprayed, e.g., through a rotating diskatomiser, into a cooling chamber where the droplets quickly solidify(Michael S. Showell (editor); Powdered detergents; Surfactant ScienceSeries; 1998; vol. 71; page 140-142; Marcel Dekker). The productobtained is one wherein the enzyme is uniformly distributed throughoutan inert material instead of being concentrated on its surface. AlsoU.S. Pat. Nos. 4,016,040 and 4,713,245 are documents relating to thistechnique;

f) mixer granulation products, wherein a liquid is added to a dry powdercomposition of, e.g., conventional granulating components, the enzymebeing introduced either via the liquid or the powder or both. The liquidand the powder are mixed and as the moisture of the liquid is absorbedin the dry powder, the components of the dry powder will start to adhereand agglomerate and particles will build up, forming granulatescomprising the enzyme. Such a process is described in U.S. Pat. No.4,106,991 and related documents EP 170360, EP 304332, EP 304331, WO90/09440 and WO 90/09428. In a particular product of this processwherein various high-shear mixers can be used as granulators, granulatesconsisting of enzyme as enzyme, fillers and binders etc. are mixed withcellulose fibres to reinforce the particles to give the so-calledT-granulate. Reinforced particles, being more robust, release lessenzymatic dust.

g) size reduction, wherein the cores are produced by milling or crushingof larger particles, pellets, tablets, briquettes etc. containing theenzyme. The wanted core particle fraction is obtained by sieving themilled or crushed product. Over and undersized particles can berecycled. Size reduction is described in (Martin Rhodes (editor);Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons);

h) fluid bed granulation, which involves suspending particulates in anair stream and spraying a liquid onto the fluidized particles vianozzles. Particles hit by spray droplets get wetted and become tacky.The tacky particles collide with other particles and adhere to them andform a granule;

i) the cores may be subjected to drying, such as in a fluid bed drier.Other known methods for drying granules in the feed or detergentindustry can be used by the skilled person. The drying preferably takesplace at a product temperature of from 25 to 90° C. For some enzymes itis important the cores comprising the enzyme contain a low amount ofwater before coating. If water sensitive enzymes are coated beforeexcessive water is removed, it will be trapped within the core and itmay affect the activity of the enzyme negatively. After drying, thecores preferably contain 0.1-10% w/w water.

The core may include additional materials such as fillers, fibrematerials (cellulose or synthetic fibres), stabilizing agents,solubilizing agents, suspension agents, viscosity regulating agents,light spheres, plasticizers, salts, lubricants and fragrances.

The core may include a binder, such as synthetic polymer, wax, fat, orcarbohydrate.

The core may include a salt of a multivalent cation, a reducing agent,an antioxidant, a peroxide decomposing catalyst and/or an acidic buffercomponent, typically as a homogenous blend.

In one embodiment, the core comprises a material selected from the groupconsisting of salts (such as calcium acetate, calcium benzoate, calciumcarbonate, calcium chloride, calcium citrate, calcium sorbate, calciumsulfate, potassium acetate, potassium benzoate, potassium carbonate,potassium chloride, potassium citrate, potassium sorbate, potassiumsulfate, sodium acetate, sodium benzoate, sodium carbonate, sodiumchloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate,zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zincsulfate), starch or a sugar or sugar derivative (such as e.g. sucrose,dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such ase.g. sucrose, dextrin, glucose, lactose, sorbitol), small organicmolecules, starch, flour, cellulose and minerals and clay minerals (alsoknown as hydrous aluminium phyllosilicates). In one embodiment, the corecomprises a clay mineral such as kaolinite or kaolin.

The core may include an inert particle with the enzyme absorbed into it,or applied onto the surface, e.g., by fluid bed coating.

The core may have a diameter of 20-2000 μm, particularly 50-1500 μm,100-1500 μm or 250-1200 μm.

The core may be surrounded by at least one coating, e.g., to improve thestorage stability, to reduce dust formation during handling, or forcoloring the granule. The optional coating(s) may include a salt and/orwax and/or flour coating, or other suitable coating materials.

The coating may be applied in an amount of at least 0.1% by weight ofthe core, e.g., at least 0.5%, 1% or 5%. The amount may be at most 100%,70%, 50%, 40% or 30%.

The coating is preferably at least 0.1 μm thick, particularly at least0.5 μm, at least 1 μm or at least 5 μm. In some embodiments thethickness of the coating is below 100 μm, such as below 60 μm, or below40 μm.

The coating should encapsulate the core unit by forming a substantiallycontinuous layer.

A substantially continuous layer is to be understood as a coating havingfew or no holes, so that the core unit is encapsulated or enclosed withfew or no uncoated areas. The layer or coating should in particular behomogeneous in thickness.

The coating can further contain other materials as known in the art,e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/orbinders, such as titanium dioxide, kaolin, calcium carbonate or talc.

A salt coating may comprise at least 60% by weight of a salt, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% by weight.

The salt may be added from a salt solution where the salt is completelydissolved or from a salt suspension wherein the fine particles are lessthan 50 μm, such as less than 10 μm or less than 5 μm.

The salt coating may comprise a single salt or a mixture of two or moresalts. The salt may be water soluble, in particular having a solubilityat least 0.1 g in 100 g of water at 20° C., preferably at least 0.5 gper 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 gper 100 g water.

The salt may be an inorganic salt, e.g., salts of sulfate, sulfite,phosphate, phosphonate, nitrate, chloride or carbonate or salts ofsimple organic acids (less than 10 carbon atoms, e.g., 6 or less carbonatoms) such as citrate, malonate or acetate. Examples of cations inthese salts are alkali or earth alkali metal ions, the ammonium ion ormetal ions of the first transition series, such as sodium, potassium,magnesium, calcium, zinc or aluminium. Examples of anions includechloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate,phosphate, monobasic phosphate, dibasic phosphate, hypophosphite,dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate,metasilicate, citrate, malate, maleate, malonate, succinate, sorbate,lactate, formate, acetate, butyrate, propionate, benzoate, tartrate,ascorbate or gluconate. In particular alkali- or earth alkali metalsalts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride orcarbonate or salts of simple organic acids such as citrate, malonate oracetate may be used.

The salt in the coating may have a constant humidity at 20° C. above60%, particularly above 70%, above 80% or above 85%, or it may beanother hydrate form of such a salt (e.g., anhydrate). The salt coatingmay be as described in WO1997/05245, WO1998/54980, WO1998/55599,WO2000/70034, WO2006/034710, WO2008/017661, WO2008/017659,WO2000/020569, WO2001/004279, WO1997/05245, WO2000/01793, WO2003/059086,WO2003/059087, WO2007/031483, WO2007/031485, WO2007/044968,WO2013/192043, WO2014/014647 and WO2015/197719 or polymer coating suchas described in WO 2001/00042.

Specific examples of suitable salts are NaCl (CH20° C.=76%), Na2CO3(CH20° C.=92%), NaNO3 (CH20° C.=73%), Na2HPO4 (CH20° C.=95%), Na3PO4(CH25° C.=92%), NH4Cl (CH20° C.=79.5%), (NH4)2HPO4 (CH20° C.=93%),NH4H2PO4 (CH20° C.=93.1%), (NH4)2SO4 (CH20° C.=81.1%), KCl (CH20°C.=85%), K2HPO4 (CH20° C.=92%), KH2PO4 (CH20° C.=96.5%), KNO3 (CH20°C.=93.5%), Na2SO4 (CH20° C.=93%), K2SO4 (CH20° C.=98%), KHSO4 (CH20°C.=86%), MgSO4 (CH20° C.=90%), ZnSO4 (CH20° C.=90%) and sodium citrate(CH25° C.=86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4,Mg(NO3)2, magnesium acetate, calcium acetate, calcium benzoate, calciumcarbonate, calcium chloride, calcium citrate, calcium sorbate, calciumsulfate, potassium acetate, potassium benzoate, potassium carbonate,potassium chloride, potassium citrate, potassium sorbate, sodiumacetate, sodium benzoate, sodium citrate, sodium sulfate, zinc acetate,zinc benzoate, zinc carbonate, zinc chloride, zinc citrate and zincsorbate.

The salt may be in anhydrous form, or it may be a hydrated salt, i.e. acrystalline salt hydrate with bound water(s) of crystallization, such asdescribed in WO 99/32595. Specific examples include anhydrous sodiumsulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfateheptahydrate (MgSO4.7H20), zinc sulfate heptahydrate (ZnSO4.7H20),sodium phosphate dibasic heptahydrate (Na2HPO4.7H20), magnesium nitratehexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesiumacetate tetrahydrate.

Preferably the salt is applied as a solution of the salt, e.g., using afluid bed.

A wax coating may comprise at least 60% by weight of a wax, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% by weight.

Specific examples of waxes are polyethylene glycols; polypropylenes;Carnauba wax; Candelilla wax; bees wax; hydrogenated plant oil or animaltallow such as polyethylene glycol (PEG), methyl hydroxy-propylcellulose (MHPC), polyvinyl alcohol (PVA), hydrogenated ox tallow,hydrogenated palm oil, hydrogenated cotton seeds and/or hydrogenated soybean oil; fatty acid alcohols; mono-glycerides and/or di-glycerides,such as glyceryl stearate, wherein stearate is a mixture of stearic andpalmitic acid; micro-crystalline wax; paraffin's; and fatty acids, suchas hydrogenated linear long chained fatty acids and derivatives thereof.A preferred wax is palm oil or hydrogenated palm oil.

The granule may comprise a core comprising the alpha-galactosidase ofthe invention, one or more salt coatings and one or more wax coatings.Examples of enzyme granules with multiple coatings are shown inWO1993/07263, WO1997/23606 and WO2016/149636.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. The coating materials can be waxy coating materialsand film-forming coating materials. Examples of waxy coating materialsare poly(ethylene oxide) products (polyethyleneglycol, PEG) with meanmolar weights of 1000 to 20000; ethoxylated nonylphenols having from 16to 50 ethylene oxide units; ethoxylated fatty alcohols in which thealcohol contains from 12 to 20 carbon atoms and in which there are 15 to80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di-and triglycerides of fatty acids. Examples of film-forming coatingmaterials suitable for application by fluid bed techniques are given inGB 1483591.

The granulate may further comprise one or more additional enzymes. Eachenzyme will then be present in more granules securing a more uniformdistribution of the enzymes, and also reduces the physical segregationof different enzymes due to different particle sizes. Methods forproducing multi-enzyme co-granulates is disclosed in the ip.comdisclosure IPCOM000200739D.

Another example of formulation of enzymes by the use of co-granulates isdisclosed in WO 2013/188331.

The present invention also relates to protected enzymes preparedaccording to the method disclosed in EP 238,216.

Thus, in a further aspect, the present invention provides a granule,which comprises:

(a) a core comprising an alpha-galactosidase according to the invention,and

(b) a coating consisting of one or more layer(s) surrounding the core.

In one embodiment, the coating comprises a salt coating as describedherein. In one embodiment, the coating comprises a wax coating asdescribed herein. In one embodiment, the coating comprises a saltcoating followed by a wax coating as described herein.

Plant Based Material

In an embodiment, the plant based material is from the taxonomicsubclass rosids such as the taxonomic order Fabales or the the taxonomicorder Brassicales.

In one embodiment, the plant based material from is from the familyFabaceae, such as the subfamilies Caesalpinioideae or Mimosoideae orPapilionoideae. In an embodiment, the plant based material from is fromthe sub-family Papilionoideae, such as the tribe Abreae orAmorpheae orBossiaeeae or Brongniartieae or Cicereae or Crotalarieae or Dalbergieaeor Desmodieae or Dipterygeae or Euchresteae or Fabeae or Galegeae orGenisteae or Hedysareae or Hypocalypteae or Indigofereae or Loteae orMillettieae or Mirbelieae or Phaseoleae or Podalyrieae or Psoraleeae orRobinieae or Sesbanieae or Sophoreae or Swartzieae or Thermopsideae orTrifolieae.

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the tribe Phaseoleae, such as the genusAdenodolichos or Alistilus or Amphicarpaea or Ancistrotropis or Apios orAtylosia or Bionia or Bolusafra or Butea or Cajanus or Calopogonium orCamptosema or Canavalia or Centrosema or Cleobulia or Clitoria orCochlianthus or Cochliasanthus or Collaea or Cologania or Condylostylisor Cratylia or Cymbosema or Decorsea or Dioclea or Dipogon orDolichopsis or Dolichos or Dumasia or Dunbaria or Eriosema or Erythrinaor Flemingia or Galactia or Glycine or Hardenbergia or Helicotropis orKennedia or Lablab or Leptospron or Macroptilium or Macrotyloma orMastersia or Mucuna or Mysanthus or Neonotonia or Neorautanenia orNesphostylis or Nogra or Ophrestia or Otoptera or Oxyrhynchus orPachyrhizus or Paracalyx or Phaseolus or Phylacium or Physostigma orPseudeminia or Pseudovigna or Psophocarpus or Pueraria or Ramirezella orRhodopis or Rhynchosia or Shuteria or Sigmoidotropis or Sinodolichos orSpathionema or Spatholobus or Sphenostylis or Strongylodon orStrophostyles or Teramnus or Teyleria or Vandasina or Vatovaea or Vignaor Wajira.

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the genus Glycine, such as the species Glycineaff. tabacina or Glycine albicans or Glycine aphyonota or Glycinearenaria or Glycine argyrea or Glycine canescens or Glycine clandestinaor Glycine curvata or Glycine cyrtoloba or Glycine dolichocarpa orGlycine falcata or Glycine gracei or Glycine hirticaulis or Glycinelactovirens or Glycine latifolia or Glycine latrobeana or Glycinemicrophylla or Glycine peratosa or Glycine pindanica or Glycine pulleniior Glycine rubiginosa or Glycine stenophita or Glycine syndetika orGlycine tabacina or Glycine tomentella or Glycine sp. T1 or Glycine sp.T5 or Glycine gracilis or Glycine max (soy bean) or Glycine max xGlycine soja or Glycine soja (wild soybean).

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the genus Cajanus such as the species Cajanuscajan (pigeon pea), Cajanus cajanifolius and Cajanus scarabaeoide.

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the genus Phaseolus, such as the speciesPhaseolus acutifolius (tepary bean) or Phaseolus acutifolius var.latifolius or Phaseolus albescens or Phaseolus albiflorus or Phaseolusalbinervus or Phaseolus altimontanus or Phaseolus amblyosepalus orPhaseolus angustissimus or Phaseolus augusti or Phaseolus bolivianus orPhaseolus campanulatus or Phaseolus carteri or Phaseolus chiapasanus orPhaseolus coccineus (scarlet runner bean) or Phaseolus coccineus subsp.coccineus or Phaseolus coccineus subsp. polyanthus or Phaseoluscostaricensis or Phaseolus dasycarpus or Phaseolus dumosus or Phaseolusesperanzae or Phaseolus esquincensis or Phaseolus filiformis (slimjimbean) or Phaseolus glabellus or Phaseolus gladiolatus or Phaseolusgrayanus or Phaseolus hintonii or Phaseolus jaliscanus or Phaseolusjuquilensis or Phaseolus laxiflorus or Phaseolus leptostachyus orPhaseolus lignosus or Phaseolus lunatus (lima bean) or Phaseolusmacrolepis or Phaseolus maculatifolius or Phaseolus maculatus (cocolmecabean) or Phaseolus maculatus subsp. ritensis or Phaseolus macvaughii orPhaseolus magnilobatus or Phaseolus marechalii or Phaseolus micranthusor Phaseolus microcarpus or Phaseolus mollis or Phaseolus neglectus orPhaseolus nelsonii or Phaseolus nodosus or Phaseolus novoleonensis orPhaseolus oaxacanus or Phaseolus oligospermus or Phaseoluspachyrrhizoides or Phaseolus parvifolius or Phaseolus parvulus orPhaseolus pauciflorus or Phaseolus pedicellatus or Phaseolus perplexusor Phaseolus persistentus or Phaseolus plagiocylix or Phaseoluspluriflorus or Phaseolus polymorphus or Phaseolus polystachios orPhaseolus polystachios subsp. sinuatus or Phaseolus polystachios subsp.smilacifolius or Phaseolus reticulatus or Phaseolus rotundatus orPhaseolus salicifolius or Phaseolus sonorensis or Phaseolustalamancensis or Phaseolus tenellus or Phaseolus texensis or Phaseolustuerckheimii or Phaseolus vulgaris (French bean) or Phaseolus vulgarisvar. aborigineus or Phaseolus vulgaris var. nanus or Phaseolusxanthotrichus or Phaseolus xolocotzii or Phaseolus zimapanensis.

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the tribe Cicereae, such as the genus Cicer, suchas the species Cicer anatolicum or Cicer arietinum (chickpea) or Cicerbijugum or Cicer canariense or Cicer chorassanicum or Cicer cuneatum orCicer echinospermum or Cicer flexuosum or Cicer floribundum or Cicergraecum or Cicer incisum or Cicer isauricum or Cicerjudaicum or Cicerkermanense or Cicer macracanthum or Cicer microphyllum or Cicermontbretii or Cicer multijugum or Cicer nuristanicum or Cicer oxyodon orCicer pinnatifidum or Cicer pungens or Cicer rechingeri or Cicerreticulatum or Cicer songaricum or Cicer spiroceras or Cicer stapfianumor Cicer subaphyllum or Cicer tragacanthoides or Cicer yamashitae

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the tribe Genisteae, such as the genusAdenocarpus or Anarthrophyllum or Argyrocytisus or Argyrolobium orCalicotome or Chamaecytisus or Cytisophyllum or Cytisus or Dichilus orEchinospartum or Erinacea or Genista or Gonocytisus or Hesperolaburnumor Laburnum or Lembotropis or Lupinus or Melolobium or Petteria orPodocytisus or Polhillia or Retama or Sellocharis or Spartium orStauracanthus or Teline or Ulex

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the tribe Fabeae, such as the genus Lathyrus orLens or Pisum or Vavilovia or Vicia. In one embodiment, the plant basedmaterial from the sub-family Papilionoideae is from the genus Lens, suchas the species Lens culinaris (lentil) or Lens culinaris subsp.culinaris or Lens culinaris subsp. odemensis or Lens culinaris subsp.tomentosus or Lens cyanea or Lens ervoides or Lens lamottei or Lensnigricans or Lens orientalis (ye bing dou).

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the genus Vicia, such as the species Viciagarinensis or Vicia sojakii or Vicia rechingeri or Vicia kurdica orVicia multijuga or Vicia akhmaganica or Vicia variabilis or Viciavariegata or Vicia persica or Vicia kotschyana or Vicia hirta or Viciagregaria or Vicia ciceroidea or Vicia cappadocica or Vicia balansae orVicia aucheri or Vicia sp. ‘telaponensis’ or Vicia venulosa or Viciasubvillosa or Vicia stenophylla or Vicia sicula or Vicia sibthorpii orVicia semiglabra or Vicia scandens or Vicia pinetorum or Vicia picta orVicia pectinata or Vicia paucifolia or Vicia palaestina or Viciaonobrychioides or Vicia ochroleuca or Vicia nataliae or Viciamontevidensis or Vicia monardii or Vicia minutiflora or Vicia menziesiior Vicia megalotropis or Vicia malosana or Vicia lunata or Vicialeucantha or Vicia leavenworthii or Vicia larissae or Vicia iranica orVicia incana or Vicia hololasia or Vicia glauca or Vicia freyniana orVicia floridana or Vicia filicaulis or Vicia ferreirensis or Viciaexigua or Vicia dennesiana or Vicia cypria or Vicia cretica or Viciacostata or Vicia claessensii or Vicia chaetocalyx or Vicia cassia orVicia capreolata or Vicia caesarea or Vicia biennis or Vicia baicalensisor Vicia altissima or Vicia alpestris or Vicia acutifolia or Viciapubescens or Vicia cirrhosa or Vicia koeieana or Vicia ramuliflora orVicia multicaulis or Vicia parviflora or Vicia vicioides or Viciatenuifolia or Vicia orobus or Vicia nigra or Vicia incisa or Viciaepetiolaris or Vicia crocea or Vicia sparsiflora or Vicia nummularia orVicia dichroantha or Vicia cassubica or Vicia monantha (bard vetch) orVicia cinerea or Vicia oroboides or Vicia tibetica or Vicia caroliniana(Carolina or wood vetch) or Vicia disperma or Vicia esdraelonensis orVicia pulchella or Vicia mexicana or Vicia leucophaea or Vicia humilisor Vicia barbazitae or Vicia pyrenaica or Vicia qatmensis or Vicialathyroides or Vicia cuspidata or Vicia dionysiensis or Vicia abbreviataor Vicia sepium or Vicia sericocarpa or Vicia noeana or Vicia hyrcanicaor Vicia hybrida or Vicia galeata or Vicia ciliatula or Vicia assyriacaor Vicia tigridis or Vicia anatolica or Vicia sylvatica or Viciadumetorum or Vicia mollis or Vicia aintabensis or Vicia peregrina orVicia lutea (yellow vetch) or Vicia grandiflora or Vicia articulata orVicia americana or Vicia michauxii or Vicia vicina or Vicia venosa orVicia tetrasperma or Vicia ervilia or Vicia benghalensis (purple orwinter vetch) or Vicia angustipinnata or Vicia amurensis or Viciaunijuga or Vicia pseudo-orobus or Vicia pisiformis or Vicia nipponica orVicia nigricans or Vicia linearifolia or Vicia japonica or Viciahirticalycina or Vicia fauriae or Vicia chosenensis or Vicia bungei orVicia bifolia or Vicia amoena or Vicia montbretii or Vicia serratifoliaor Vicia paucijuga or Vicia kalakhensis or Vicia johannis or Viciahyaeniscyamus or Vicia galilaea or Vicia eristalioides or Viciabithynica or Vicia melanops or Vicia ludoviciana or Vicia pannonica orVicia narbonensis or Vicia villosa or Vicia hirsuta or Vicia sativa(spring vetch) or Vicia faba (broad bean or fava bean) or Vicia cracca(bird vetech).

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the genus Pisum, such as the species Pisumabyssinicum (Abyssinian pea) or Pisum fulvum or Pisum sativum (pea) orPisum sativum subsp. asiaticum or Pisum sativum subsp. elatius (wildpea) or Pisum sativum var. pumilio (Syrian fodder pea) or Pisum sativumsubsp. jomardii or Pisum sativum subsp. Sativum or Pisum sativum var.arvense or Pisum sativum var. choresmicum or Pisum sativum var.macrocarpon (snow pea) or Pisum sativum var. ponderosum or Pisum sativumvar. tibetanicum or Pisum sativum subsp. transcaucasicum

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the tribe Dalbergieae, such as the genus Adesmiaor Aeschynomene or Amicia or Andira or Arachis or Brya or Bryaspis orCascaronia or Centrolobium or Chaetocalyx or Chapmannia or Cranocarpusor Cyclocarpa or Dalbergia or Diphysa or Discolobium or Etaballia orFiebrigiella or Fissicalyx or Geissaspis or Geoffroea or Grazielodendronor Humularia or Hymenolobium or Inocarpus or Kotschya or Machaerium orMaraniona or Nissolia or Ormocarpopsis or Ormocarpum or Paramachaeriumor Peltiera or Pictetia or Platymiscium or Platypodium or Poiretia orPterocarpus or Ramorinoa or Riedeliella or Smithia or Soemmeringia orSteinbachiella or Stylosanthes or Tipuana or Weberbauerella or Zornia.

In one embodiment, the plant based material from the sub-familyPapilionoideae is from the genus Arachis, such as the speciesAppressipila (amendoim bravo) or Arachis batizocoi or Arachisbrevipetiolata or Arachis burchellii or Arachis burkartii or Arachiscardenasii or Arachis chiquitana or Arachis correntina or Arachiscruziana or Arachis decora or Arachis diogoi or Arachis duranensis orArachis duranensis x Arachis stenosperma or Arachis glabrata(amendoim-bravo) or Arachis glabrata var. glabrata or Arachis glabratavar. hagenbeckii or Arachis glabrata x Arachis hypogaea or Arachisglandulifera or Arachis guaranitica or Arachis helodes or Arachishermannii or Arachis hoehnei or Arachis hypogaea (peanut) or Arachishypogaea subsp. Fastigiata or Arachis hypogaea var. vulgaris (Spanishpeanut) or Arachis hypogaea subsp. Hypogaea or Arachis hypogaea var.hirsuta or Arachis ipaensis or Arachis ipaensis x Arachis magna orArachis kempff-mercadoi or Arachis kretschmeri or Arachis kuhlmannii orArachis linearifolia or Arachis lutescens or Arachis magna or Arachismajor or Arachis matiensis or Arachis microsperma or Arachis monticolaor Arachis palustris or Arachis paraguariensis or Arachis paraguariensissubsp. capibarensis or Arachis paraguariensis subsp. paraguariensis orArachis pflugeae or Arachis pintoi or Arachis praecox or Arachis pusilla(amendoim de caracar) or Arachis repens or Arachis rigonii or Arachisschinini or Arachis simpsonii or Arachis stenophylla or Arachisstenosperma or Arachis stenosperma x Arachis cardenasii or Arachissylvestris (amendoim do porco) or Arachis trinitensis or Arachistriseminata or Arachis tuberosa or Arachis valida or Arachis villosa orArachis villosulicarpa or Arachis williamsii.

In one aspect, the plant based material is from the taxonomic orderBrassicales, such as the family Brassicaceae, preferably the tribeBrassiceae, more preferably the family Brassica.

In one embodiment, the plant based material from the tribe Brassiceae isfrom the family Brassica, such as Brassica aucheri, Brassica balearica,Brassica barrelieri, Brassica carinata (Abyssinian mustard), Brassicacarinata x Brassica napus, Brassica carinata x Brassica rapa, Brassicacretica, Brassica deflexa, Brassica desnottesii, Brassica drepanensis,Brassica elongata, Brassica fruticulosa, Brassica fruticulosa subsp.cossoniana, Brassica fruticulosa subsp. mauritanica, Brassicafruticulosa subsp. rifana, Brassica gravinae, Brassica hilarionis,Brassica hybrid cultivar, Brassica incana, Brassica insularis, Brassicainsularis subsp. insularis, Brassica juncea (Indian mustard), Brassicajuncea var. crassicaulis, Brassica juncea var. gemmifera, Brassicajuncea var. gracilis, Brassica juncea var. juncea, Brassica juncea var.multiceps, Brassica juncea var. multisecta, Brassica juncea var.napiformis (jie cai ge da), Brassica juncea var. rugosa, Brassica junceavar. strumata, Brassica juncea var. subintegrifolia, Brassica junceavar. tumida (zha cai), Brassica juncea var. utilis, Brassica macrocarpa,Brassica maurorum, Brassica montana, Brassica napus (rape), Brassicanapus subsp. rapifera (Swedish turnip), Brassica napus var. napus(annual rape), Brassica napus x Brassica rapa, Brassica nigra (blackmustard), Brassica nigra var. abyssinica, Brassica oleracea, Brassicaoleracea var. albiflora, Brassica oleracea var. alboglabra (Chinesekale), Brassica oleracea var. botrytis (cauliflower), Brassica oleraceavar. capitata (cabbage), Brassica oleracea var. costata (Bedfordcabbage), Brassica oleracea var. gemmifera (Brussels sprouts), Brassicaoleracea var. gongylodes (kohlrabi), Brassica oleracea var. italica(asparagus broccoli), Brassica oleracea var. medullosa (marrow-stemkale), Brassica oleracea var. oleracea, Brassica oleracea var. ramosa(branching bush kale), Brassica oleracea var. sabauda, Brassica oleraceavar. viridis (kale), Brassica oleracea x Brassica rapa subsp.pekinensis, Brassica oxyrrhina, Brassica procumbens, Brassica rapa(field mustard), Brassica rapa subsp. chinensis (bok-choy), Brassicarapa var. parachinensis (cai xin), Brassica rapa var. purpuraria (purplestem mustard), Brassica rapa subsp. narinosa, Brassica rapa subsp.nipposinica (mizuna), Brassica rapa var. perviridis (kabuna), Brassicarapa subsp. oleifera (biennial turnip rape), Brassica rapa(Nippo-oleifera Group), Brassica rapa subsp. pekinensis (Chinesecabbage), Brassica rapa subsp. rapa (turnip), Brassica rapa var.oleifera, Brassica rapa x Brassica nigra, Brassica repanda, Brassicarepanda subsp. baldensis, Brassica repanda subsp. blancoana, Brassicarepanda subsp. cadevallii, Brassica repanda subsp. confusa, Brassicarepanda subsp. glabrescens, Brassica repanda subsp. gypsicola, Brassicarepanda subsp. latisiliqua, Brassica repanda subsp. maritima, Brassicarepanda subsp. repanda, Brassica repanda subsp. saxatilis, Brassicarupestris, Brassica ruvo (broccoletto), Brassica souliei, Brassicasouliei subsp. amplexicaulis, Brassica spinescens, Brassicatournefortii, Brassica villosa or Brassica villosa subsp. Bivoniana.

In particular embodiments, the plant based material is soybean, wildsoybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean,lima bean, French bean, Broad bean (fava bean), chickpea, lentil,peanut, Spanish peanut, canola, rapeseed (oilseed rape) or pea or in aprocessed form such as soybean meal, full fat soy bean meal, soy proteinconcentrate (SPC), fermented soybean meal (FSBM) or any combinationthereof. In a preferred embodiment, the plant based material is soybeanor soybean meal.

Animal Feed and Animal Feed Additives

The present invention also relates to animal feed compositions andanimal feed additives comprising one or more alpha-galactosidases of theinvention. In an embodiment, the animal feed or animal feed additivecomprises a formulating agent and one or more alpha-galactosidases ofthe invention. In a further embodiment, the formulating agent comprisesone or more of the following compounds: glycerol, ethylene glycol, 1,2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodiumbenzoate, potassium sorbate, sodium sulfate, potassium sulfate,magnesium sulfate, sodium thiosulfate, calcium carbonate, sodiumcitrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolinand cellulose.

Animal feed compositions or diets have a relatively high content ofprotein. Poultry and pig diets can be characterised as indicated inTable B of WO 01/58275, columns 2-3. Fish diets can be characterised asindicated in column 4 of this Table B. Furthermore such fish dietsusually have a crude fat content of 200-310 g/kg.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least onealpha-galactosidase as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e. Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen by, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein as defined above.

The animal feed composition of the invention may also contain animalprotein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal,typically in an amount of 0-25%. The animal feed composition of theinvention may also comprise Dried Distillers Grains with Solubles(DDGS), typically in amounts of 0-30%.

The animal feed composition of the invention may also contain insectprotein, such as protein from mealworm, housefly or black soldier flylarvae, typically in meal form. Insect meal may replace fishmealentirely or in part, and thus may constitute 0-10% of the total feed.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

The animal feed may comprise vegetable proteins. In particularembodiments, the protein content of the vegetable proteins is at least10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may bederived from vegetable protein sources, such as legumes and cereals, forexample, materials from plants of the families Fabaceae (Leguminosae),Brassicaceae, Amaranthaceae, and Poaceae, such as soybean meal, lupinmeal, rapeseed meal, and combinations thereof.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g., soybean, lupine,pea, or bean. In another particular embodiment, the vegetable proteinsource is material from one or more plants of the family Amaranthaceae,e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetableprotein sources are rapeseed, crambe and cabbage. In another particularembodiment, soybean is a preferred vegetable protein source. Otherexamples of vegetable protein sources are cereals such as barley, wheat,rye, oat, maize (corn), rice, and sorghum.

Animal diets can e.g. be manufactured as mash feed (non-pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Enzymes canbe added as solid or liquid enzyme formulations. For example, for mashfeed a solid or liquid enzyme formulation may be added before or duringthe ingredient mixing step. For pelleted feed the (liquid or solid)alpha-galactosidase/enzyme preparation may also be added before orduring the feed ingredient step. Typically a liquidalpha-galactosidase/enzyme preparation comprises the alpha-galactosidaseof the invention optionally with a polyol, such as glycerol, ethyleneglycol or propylene glycol, and is added after the pelleting step, suchas by spraying the liquid formulation onto the pellets. The enzyme mayalso be incorporated in a feed additive or premix.

Alternatively, the alpha-galactosidase can be prepared by freezing amixture of liquid enzyme solution with a bulking agent such as groundsoybean meal, and then lyophilizing the mixture.

In an embodiment, the animal feed or animal feed additive comprises oneor more additional enzymes. In an embodiment, the animal feed comprisesone or more microbes. In an embodiment, the animal feed comprises one ormore vitamins. In an embodiment, the animal feed comprises one or moreminerals. In an embodiment, the animal feed comprises one or more aminoacids. In an embodiment, the animal feed comprises one or more otherfeed ingredients.

In another embodiment, the animal feed or animal feed additive comprisesthe polypeptide of the invention, one or more formulating agents and oneor more additional enzymes. In an embodiment, the animal feed or animalfeed additive comprises the polypeptide of the invention, one or moreformulating agents and one or more microbes. In an embodiment, theanimal feed comprises the polypeptide of the invention, one or moreformulating agents and one or more vitamins. In an embodiment, theanimal feed or animal feed additive comprises one or more minerals. Inan embodiment, the animal feed or animal feed additive comprises thepolypeptide of the invention, one or more formulating agents and one ormore amino acids. In an embodiment, the animal feed or animal feedadditive comprises the polypeptide of the invention, one or moreformulating agents and one or more other feed ingredients.

In a further embodiment, the animal feed or animal feed additivecomprises the polypeptide of the invention, one or more formulatingagents and one or more components selected from the list consisting of:one or more additional enzymes; one or more microbes; one or morevitamins; one or more minerals; one or more amino acids; and one or moreother feed ingredients.

In an embodiment, the animal feed additive comprises one or moreformulating agents, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or moreadditional enzymes, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or moreprobiotics, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or morevitamins, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or moreminerals, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or more aminoacids, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or moreprebiotics, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or more organicacids, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or morephytogenics, preferably as described herein below.

The final enzyme concentration in the diet is within the range of0.01-200 mg enzyme protein per kg diet, preferably between 0.1-100 mg/kgdiet, more preferably 0.5-50 mg, even more preferably 1-25 mg enzymeprotein per kg animal diet.

It is at present contemplated that the enzyme is administered in one ormore of the following amounts (dosage ranges): 0.01-200; 0.05-100;0.1-50; 0.2-20; 0.1-1; 0.2-2; 0.5-5; 5-50; 10-30; 20-30; 10-20; 15-25;or 1-10; —all these ranges being in mg alpha-galactosidase protein perkg feed (ppm).

For determining mg alpha-galactosidase protein per kg feed, thealpha-galactosidase is purified from the feed composition, and thespecific activity of the purified alpha-galactosidase is determinedusing a relevant assay (see under alpha-galactosidase activity). Thealpha-galactosidase activity of the feed composition as such is alsodetermined using the same assay, and on the basis of these twodeterminations, the dosage in mg alpha-galactosidase protein per kg feedis calculated.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The same principles apply for determining mg alpha-galactosidase proteinin feed additives. Of course, if a sample is available of thealpha-galactosidase used for preparing the feed additive or the feed,the specific activity is determined from this sample (no need to purifythe alpha-galactosidase from the feed composition or the additive).

Additional Enzymes

In another embodiment, the compositions described herein optionallyinclude one or more enzymes. Enzymes can be classified on the basis ofthe handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also theENZYME site at the internet: http://www.expasy.ch/enzyme/. ENZYME is arepository of information relative to the nomenclature of enzymes. It isprimarily based on the recommendations of the Nomenclature Committee ofthe International Union of Biochemistry and Molecular Biology (IUB-MB),Academic Press, Inc., 1992, and it describes each type of characterizedenzyme for which an EC (Enzyme Commission) number has been provided(Bairoch A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305).This IUB-MB Enzyme nomenclature is based on their substrate specificityand occasionally on their molecular mechanism; such a classificationdoes not reflect the structural features of these enzymes.

Another classification of certain glycoside hydrolase enzymes, such asendoglucanase, galactanase, mannanase, dextranase, lysozyme andgalactosidase is described in Henrissat et al, “The carbohydrate-activeenzymes database (CAZy) in 2013”, Nucl. Acids Res. (1 Jan. 2014) 42(D1): D490-D495; see also www.cazy.org.

Thus the composition of the invention may also comprise at least oneother enzyme selected from the group comprising of acetylxylan esterase(EC 3.1.1.23), acylglycerol lipase (EC 3.1.1.72), alpha-amylase (EC3.2.1.1), beta-amylase (EC 3.2.1.2), arabinofuranosidase (EC 3.2.1.55),cellobiohydrolases (EC 3.2.1.91), cellulase (EC 3.2.1.4), feruloylesterase (EC 3.1.1.73), galactanase (EC 3.2.1.89), alpha-galactosidase(EC 3.2.1.22), beta-galactosidase (EC 3.2.1.23), beta-glucanase (EC3.2.1.6), beta-glucosidase (EC 3.2.1.21), triacylglycerol lipase (EC3.1.1.3), lysophospholipase (EC 3.1.1.5), lysozyme (EC 3.2.1.17),alpha-mannosidase (EC 3.2.1.24), beta-mannosidase (mannanase) (EC3.2.1.25), phytase (EC 3.1.3.8, EC 3.1.3.26, EC 3.1.3.72), phospholipaseA1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), phospholipase D (EC3.1.4.4), protease (EC 3.4), pullulanase (EC 3.2.1.41), pectinesterase(EC 3.1.1.11), xylanase (EC 3.2.1.8, EC 3.2.1.136), beta-xylosidase (EC3.2.1.37), or any combination thereof.

In a particular embodiment, the composition of the invention comprises agalactanase (EC 3.2.1.89) and a beta-galactosidase (EC 3.2.1.23).

In a particular embodiment, the composition of the invention comprises aphytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially availablephytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P, Ronozyme®NP and Ronozyme® HiPhos (DSM Nutritional Products), Natuphos™ (BASF),Natuphos™ E (BASF), Finase® and Quantum® Blue (AB Enzymes), OptiPhos®(Huvepharma), AveMix® Phytase (Aveve Biochem), Phyzyme® XP(Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytasesinclude those described in e.g. WO 98/28408, WO 00/43503, and WO03/066847.

In a particular embodiment, the composition of the invention comprises axylanase (EC 3.2.1.8 or EC 3.2.1.136). Examples of commerciallyavailable xylanases include Ronozyme® WX (DSM Nutritional Products),Econase® XT and Barley (AB Vista), Xylathin® (Verenium), Hostazym® X(Huvepharma), Axtra® XB (Xylanase/beta-glucanase, DuPont) and Axtra® XAP(Xylanase/amylase/protease, DuPont), AveMix® XG 10 (xylanase/glucanase)and AveMix® 02 CS (xylanase/glucanase/pectinase, Aveve Biochem), andNaturgrain (BASF).

In a particular embodiment, the composition of the invention comprises aprotease (EC 3.4). Examples of commercially available proteases includeRonozyme® ProAct (DSM Nutritional Products).

In a particular embodiment, the composition of the invention comprisesan alpha-amylase (EC 3.2.1.1). Examples of commercially availablealpha-amylases include Ronozyme® A and RONOZYME® RumiStar™ (DSMNutritional Products).

In one embodiment, the composition of the invention comprises amulticomponent enzyme product, such as FRA® Octazyme (Framelco),Ronozyme® G2, Ronozyme® VP and Ronozyme® MultiGrain (DSM NutritionalProducts), Rovabio® Excel or Rovabio® Advance (Adisseo).

Eubiotics

Eubiotics are compounds which are designed to give a healthy balance ofthe micro-flora in the gastrointestinal tract. Eubiotics cover a numberof different feed additives, such as probiotics, prebiotics, phytogenics(essential oils) and organic acids which are described in more detailbelow.

Probiotics

In an embodiment, the animal feed composition further comprises one ormore additional probiotic. In a particular embodiment, the animal feedcomposition further comprises a bacterium from one or more of thefollowing genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus,Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or anycombination thereof.

In a preferred embodiment, animal feed composition further comprises abacterium from one or more of the following strains: Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus,Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, Enterococcus faecium, Enterococcus spp,and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp,Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis,Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillusfarciminus, Lactobacillus rhamnosus, Clostridium butyricum,Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri,Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii,Propionibacteria sp.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus subtilis: 3A-P4 (PTA-6506), 15A-P4(PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01(NRRL-B-50104), BS27 (NRRL B-501 05), BS 18 (NRRL B-50633), BS 278 (NRRLB-50634), DSM 29870, DSM 29871, DSM 32315, NRRL B-50136, NRRL B-50605,NRRL B-50606, NRRL B-50622 and PTA-7547.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus pumilus: NRRL B-50016, ATCC 700385, NRRLB-50885 or NRRL B-50886.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus lichenformis: NRRL B 50015, NRRL B-50621or NRRL B-50623.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus amyloliquefaciens: DSM 29869, DSM 29869,NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B-50606, NRRLB-50013, NRRL B-50151, NRRL B-50141, NRRL B-50147 or NRRL B-50888.

The bacterial count of each of the bacterial strains in the animal feedcomposition is between 1×10⁴ and 1×10¹⁴ CFU/kg of dry matter, preferablybetween 1×10⁶ and 1×10¹² CFU/kg of dry matter, and more preferablybetween 1×10⁷ and 1×10¹¹ CFU/kg of dry matter. In a more preferredembodiment the bacterial count of each of the bacterial strains in theanimal feed composition is between 1×10⁸ and 1×10¹⁰ CFU/kg of drymatter.

The bacterial count of each of the bacterial strains in the animal feedcomposition is between 1×10⁵ and 1×10¹⁵ CFU/animal/day, preferablybetween 1×10⁷ and 1×10¹³ CFU/animal/day, and more preferably between1×10⁸ and 1×10¹² CFU/animal/day. In a more preferred embodiment thebacterial count of each of the bacterial strains in the animal feedcomposition is between 1×10⁹ and 1×10¹¹ CFU/animal/day.

In another embodiment, the one or more bacterial strains are present inthe form of a stable spore.

Examples of commercial products are Cylactin® (DSM NutritionalProducts), Alterion (Adisseo), Enviva PRO (DuPont Animal Nutrition),Gallipro®, Gallipro® Max, Probios® Guard, Lactiferm® and Bioplus® (ChrHansen), PoultryStar®, PoultryStar® sol, PoultryStar® me, AquaStar®(Biomin), Syncra® (mix enzyme+probiotic, DuPont Animal Nutrition),Ecobiol® and Fecinor® (Norel/Evonik) and GutCare® PY1 (Evonik).

Prebiotics

Prebiotics are substances that induce the growth or activity ofmicroorganisms (e.g., bacteria and fungi) that contribute to thewell-being of their host. Prebiotics are typically non-digestible fibercompounds that pass undigested through the upper part of thegastrointestinal tract and stimulate the growth or activity ofadvantageous bacteria that colonize the large bowel by acting assubstrate for them. Normally, prebiotics increase the number or activityof bifidobacteria and lactic acid bacteria in the GI tract.

Yeast derivatives (inactivated whole yeasts or yeast cell walls) canalso be considered as prebiotics. They often comprisemannan-oligosaccharids, yeast beta-glucans or protein contents and arenormally derived from the cell wall of the yeast, Saccharomycescerevisiae.

Examples of yeast products are Yang® and Agrimos (Lallemand AnimalNutrition).

Phytogenics

Phytogenics are a group of natural growth promoters or non-antibioticgrowth promoters used as feed additives, derived from herbs, spices orother plants. Phytogenics can be single substances prepared fromessential oils/extracts, essential oils/extracts, single plants andmixture of plants (herbal products) or mixture of essentialoils/extracts/plants (specialized products).

Examples of phytogenics are rosemary, sage, oregano, thyme, clove, andlemongrass. Examples of essential oils are thymol, eugenol, meta-cresol,vaniline, salicylate, resorcine, guajacol, gingerol, lavender oil,ionones, irone, eucalyptol, menthol, peppermint oil, alpha-pinene;limonene, anethol, linalool, methyl dihydrojasmonate, carvacrol,propionic acid/propionate, acetic acid/acetate, butyric acid/butyrate,rosemary oil, clove oil, geraniol, terpineol, citronellol, amyl and/orbenzyl salicylate, cinnamaldehyde, plant polyphenol (tannin), turmericand curcuma extract.

Examples of commercial products are Crina® (DSM Nutritional Products);Cinergy™, Cinergy™ FIT, Biacid™, (Cargill), Digestarom® and Digestarom®DC (Biomin) and Envivo EO (DuPont Animal Nutrition).

Organic Acids

Organic acids (C1-C7) are widely distributed in nature as normalconstituents of plants or animal tissues. They are also formed throughmicrobial fermentation of carbohydrates mainly in the large intestine.They are often used in swine and poultry production as a replacement ofantibiotic growth promoters since they have a preventive effect on theintestinal problems like necrotic enteritis in chickens and Escherichiacoli infection in young pigs. Organic acids can be sold as monocomponent or mixtures of typically 2 or 3 different organic acids.Examples of organic acids are propionic acid, formic acid, citric acid,lactic acid, sorbic acid, malic acid, acetic acid, fumaric acid, benzoicacid, butyric acid and tartaric acid or their salt (typically sodium orpotassium salt such as potassium diformate or sodium butyrate).

Examples of commercial products are VevoVitall® (DSM NutritionalProducts), Amasil®, Luprisil®, Lupro-Grain®, Lupro-Cid®, Lupro-Mix®,Lupro-Mix® NA (BASF), n-Butyric Acid AF (OXEA), Biacid™, Prohacid™Classic and Prohacid™ Advance™ (Cargill), Biotronic® (Biomin) and AdimixPrecision (Nutriad).

Premix

The incorporation of the composition of feed additives as exemplifiedherein above to animal feeds, for example poultry feeds, is in practicecarried out using a concentrate or a premix. A premix designates apreferably uniform mixture of one or more microingredients with diluentand/or carrier. Premixes are used to facilitate uniform dispersion ofmicro-ingredients in a larger mix. A premix according to the inventioncan be added to feed ingredients or to the drinking water as solids (forexample as water soluble powder) or liquids.

Amino Acids

The composition of the invention may further comprise one or more aminoacids. Examples of amino acids which are used in animal feed are lysine,alanine, beta-alanine, threonine, methionine and tryptophan.

Vitamins and Minerals

In another embodiment, the animal feed may include one or more vitamins,such as one or more fat-soluble vitamins and/or one or morewater-soluble vitamins. In another embodiment, the animal feed mayoptionally include one or more minerals, such as one or more traceminerals and/or one or more macro minerals.

Usually fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed.

Non-limiting examples of fat-soluble vitamins include vitamin A, vitaminD3, vitamin E, and vitamin K, e.g., vitamin K3.

Non-limiting examples of water-soluble vitamins include vitamin B12,biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folicacid and panthothenate, e.g., Ca-D-panthothenate.

Non-limiting examples of trace minerals include boron, cobalt, chloride,chromium, copper, fluoride, iodine, iron, manganese, molybdenum,selenium and zinc.

Non-limiting examples of macro minerals include calcium, magnesium,potassium and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

In a still further embodiment, the animal feed additive of the inventioncomprises at least one of the below vitamins, preferably to provide anin-feed-concentration within the ranges specified in the below Table 1(for piglet diets, and broiler diets, respectively).

TABLE 1 Typical vitamin recommendations Vitamin Piglet diet Broiler dietVitamin A 10,000-15,000 IU/kg feed 8-12,500 IU/kg feed Vitamin D31800-2000 IU/kg feed 3000-5000 IU/kg feed Vitamin E 60-100 mg/kg feed150-240 mg/kg feed Vitamin K3 2-4 mg/kg feed 2-4 mg/kg feed Vitamin B12-4 mg/kg feed 2-3 mg/kg feed Vitamin B2 6-10 mg/kg feed 7-9 mg/kg feedVitamin B6 4-8 mg/kg feed 3-6 mg/kg feed Vitamin B12 0.03-0.05 mg/kgfeed 0.015-0.04 mg/kg feed Niacin 30-50 mg/kg feed 50-80 mg/kg feed(Vitamin B3) Pantothenic 20-40 mg/kg feed 10-18 mg/kg feed acid Folicacid 1-2 mg/kg feed 1-2 mg/kg feed Biotin 0.15-0.4 mg/kg feed 0.15-0.3mg/kg feed Choline 200-400 mg/kg feed 300-600 mg/kg feed chlorideOther Feed Ingredients

The composition of the invention may further comprise colouring agents,stabilisers, growth improving additives and aroma compounds/flavourings,polyunsaturated fatty acids (PUFAs); reactive oxygen generating species,anti-microbial peptides and anti-fungal polypeptides.

Examples of colouring agents are carotenoids such as beta-carotene,astaxanthin, and lutein.

Examples of aroma compounds/flavourings are creosol, anethol, deca-,undeca-and/or dodeca-lactones, ionones, irone, gingerol, piperidine,propylidene phatalide, butylidene phatalide, capsaicin and tannin.

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in WO 03/044049 and WO 03/048148, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and enzymes such as an oxidase,an oxygenase or a syntethase.

The composition of the invention may further comprise at least one aminoacid. Examples of amino acids which are used in animal feed are lysine,alanine, beta-alanine, threonine, methionine and tryptophan.

Methods of Releasing Galactose

In another aspect, the invention relates to a method of releasinggalactose from plant based material, comprising treating the plant basedmaterial with the animal the animal feed or animal feed additive of thefirst aspect of the invention, the alpha-galactosidase of the secondaspect of the invention, the granule of the third aspect of theinvention or the liquid formulation of the fourth aspect of theinvention.

In one embodiment, the animal feed comprises one or more formulatingagents as defined herein. In one embodiment, the animal feed comprisesone or more additional enzymes as defined herein. In one embodiment, theanimal feed comprises one or more microbes as defined herein. In oneembodiment, the animal feed comprises plant based material from thesubclass rosids. In a preferred embodiment, the animal feed has beenpelleted.

In an embodiment, the plant based material is soybean, wild soybean,beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean,French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanishpeanut, canola, rapeseed (oilseed rape) or pea or in a processed formsuch as soybean meal, full fat soy bean meal, soy protein concentrate(SPC), fermented soybean meal (FSBM) or any combination thereof. In apreferred embodiment, the plant based material is soybean or soybeanmeal.

Methods of Improving Animal Performance

In another aspect, the invention relates to a method of improving one ormore performance parameters of an animal comprising administering to theanimal the animal feed or animal feed additive of the first aspect ofthe invention, the alpha-galactosidase of the second aspect of theinvention, the granule of the third aspect of the invention or theliquid formulation of the fourth aspect of the invention.

In a further aspect, the invention relates to a method of improving oneor more performance parameters of an animal comprising administering tothe animal the animal feed or animal feed additive of the first aspectof the invention or the alpha-galactosidase of the second aspect of theinvention and plant based material, such that the plant based materialis administered together or separately with the polypeptide of theinvention.

In one embodiment, the term ‘improving one or more performanceparameters of an animal’ means that there is an increase in body weightgain. In another embodiment, the term ‘improving one or more performanceparameters of an animal’ means that there is an improved feed conversionratio. In a further embodiment, ‘the term ‘improving one or moreperformance parameters of an animal’ means that there is an increasedfeed efficiency. In a further embodiment, the term ‘improving one ormore performance parameters of an animal’ means that there is anincrease in body weight gain and/or an improved feed conversion ratioand/or an increased feed efficiency.

In one embodiment, the animal feed comprises one or more formulatingagents as defined herein. In one embodiment, the animal feed comprisesone or more additional enzymes as defined herein. In one embodiment, theanimal feed comprises one or more microbes as defined herein. In oneembodiment, the animal feed comprises plant based material from thesubclass rosids. In a preferred embodiment, the animal feed has beenpelleted.

In an embodiment, the plant based material is soybean, wild soybean,beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean,French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanishpeanut, canola, rapeseed (oilseed rape) or pea or in a processed formsuch as soybean meal, full fat soy bean meal, soy protein concentrate(SPC), fermented soybean meal (FSBM) or any combination thereof. In apreferred embodiment, the plant based material is soybean or soybeanmeal.

Method for Improving the Nutritional Value of Animal Feed

The term improving the nutritional value of an animal feed meansimproving the availability of nutrients in the feed. The nutritionalvalues refers in particular to improving the solubilisation anddegradation of the raffinose family of oligosaccharides (RFOs), such asthe trisaccharide raffinose, the tetrasaccharide stachyose, and thepentasaccharide verbascose, thereby increasing the amount of galactosereleased which can be utilised by the animal. Consequently, an improvedgalactose release will result in an improvement of the nutritional valueof the feed, thus resulting in increased growth rate and/or weight gainand/or feed conversion (i.e. the weight of ingested feed relative toweight gain).

Thus the invention further relates to a method for improving thenutritional value of an animal feed comprising treating the animal feedwith an animal feed or animal feed additive of the first aspect of theinvention, the alpha-galactosidase of the second aspect of theinvention, the alpha-galactosidase of the second aspect of theinvention, the granule of the third aspect of the invention or theliquid formulation of the fourth aspect of the invention. In anembodiment, the animal feed will have improved nutrient digestibility.

In one embodiment, the composition comprises one or more formulatingagents as defined herein. In one embodiment, the composition comprisesone or more additional enzymes as defined herein. In one embodiment, thecomposition comprises one or more microbes as defined herein. In apreferred embodiment, the composition is a granule that optionallycomprises a salt and/or wax and/or a flour coating.

In one embodiment, the animal feed additive comprises one or moreformulating agents as defined herein. In one embodiment, the animal feedadditive comprises one or more additional enzymes as defined herein. Inone embodiment, the animal feed additive comprises one or more microbesas defined herein. In one embodiment, the animal feed additive comprisesone or more vitamins, one or more minerals and/or one or more aminoacids. In a preferred embodiment, the animal feed additive is a granulethat optionally comprises a salt and/or wax and/or a flour coating.

In an embodiment, the plant based material is from the taxonomicsubclass rosids. In one aspect, the plant based material is from thetaxonomic order Fabales, such as the family Fabaceae, preferably thesubfamilies Caesalpinioideae or Mimosoideae or Papilionoideae, or morepreferably from the tribes Phaseoleae, Cicereae, Genisteae, Fabeae,Dalbergieae or Phaseoleae. In one aspect, the plant based material isfrom the taxonomic order Brassicales, such as the family Brassicaceae,preferably the tribe Brassiceae, more preferably the family Brassica.

In particular embodiments, the plant based material is soybean, wildsoybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean,lima bean, French bean, Broad bean (fava bean), chickpea, lentil,peanut, Spanish peanut, canola, rapeseed (oilseed rape) or pea or in aprocessed form such as soybean meal, full fat soy bean meal, soy proteinconcentrate (SPC), fermented soybean meal (FSBM) or any combinationthereof. In a preferred embodiment, the plant based material is soybeanor soybean meal.

In a preferred embodiment, the animal feed has been pelleted. The animalfeed may be treated with the enzyme of the invention before thepelleting step or sprayed on after the pelleting step.

Method for Reducing the Anitnutritional Effects of an Animal Feed Anexcessive amount of oligosaccharides in the hindgut can result inantinutritional effects due to flatulence production. By reducing theamount of oligosaccharide fermentation, the antinutritional effects ofsome animal feeds can be reduced resulting in improved gut and animalhealth.

Thus the invention further relates to a method for reducing theanitnutritional effects of an animal feed comprising adding to the feedthe alpha-galactosidase of the second aspect of the invention, thegranule of the third aspect of the invention or the liquid formulationof the fourth aspect of the invention.

In one embodiment, the composition comprises one or more formulatingagents as defined herein. In one embodiment, the composition comprisesone or more additional enzymes as defined herein. In one embodiment, thecomposition comprises one or more microbes as defined herein. In apreferred embodiment, the composition is a granule that optionallycomprises a salt and/or wax and/or a flour coating.

In one embodiment, the animal feed additive comprises one or moreformulating agents as defined herein. In one embodiment, the animal feedadditive comprises one or more additional enzymes as defined herein. Inone embodiment, the animal feed additive comprises one or more microbesas defined herein. In one embodiment, the animal feed additive comprisesone or more vitamins, one or more minerals and/or one or more aminoacids. In a preferred embodiment, the animal feed additive is a granulethat optionally comprises a salt and/or wax and/or a flour coating.

In an embodiment, the plant based material is from the taxonomicsubclass rosids. In one aspect, the plant based material is from thetaxonomic order Fabales, such as the family Fabaceae, preferably thesubfamilies Caesalpinioideae or Mimosoideae or Papilionoideae, or morepreferably from the tribes Phaseoleae, Cicereae, Genisteae, Fabeae,Dalbergieae or Phaseoleae. In one aspect, the plant based material isfrom the taxonomic order Brassicales, such as the family Brassicaceae,preferably the tribe Brassiceae, more preferably the family Brassica.

In particular embodiments, the plant based material is soybean, wildsoybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean,lima bean, French bean, Broad bean (fava bean), chickpea, lentil,peanut, Spanish peanut, canola, rapeseed (oilseed rape) or pea or in aprocessed form such as soybean meal, full fat soy bean meal, soy proteinconcentrate (SPC), fermented soybean meal (FSBM) or any combinationthereof. In a preferred embodiment, the plant based material is soybeanor soybean meal.

In a preferred embodiment, the animal feed has been pelleted. The animalfeed may be treated with the enzyme of the invention before thepelleting step or sprayed on after the pelleting step.

Methods of Preparing an Animal Feed

In another aspect, the invention relates to a method of preparing ananimal feed, comprising mixing the animal feed additive of the firstaspect of the invention, the alpha-galactosidase of the second aspect ofthe invention, the granule of the third aspect of the invention or theliquid formulation of the fourth aspect of the invention with plantbased material.

In one embodiment, the composition comprises one or more formulatingagents as defined herein. In one embodiment, the composition comprisesone or more additional enzymes as defined herein. In one embodiment, thecomposition comprises one or more microbes as defined herein. In apreferred embodiment, the composition is a granule that optionallycomprises a salt and/or wax and/or a flour coating.

In one embodiment, the animal feed additive comprises one or moreformulating agents as defined herein. In one embodiment, the animal feedadditive comprises one or more additional enzymes as defined herein. Inone embodiment, the animal feed additive comprises one or more microbesas defined herein. In one embodiment, the animal feed additive comprisesone or more vitamins, one or more minerals and/or one or more aminoacids. In a preferred embodiment, the animal feed additive is a granulethat optionally comprises a salt and/or wax and/or a flour coating.

In an embodiment, the plant based material is from the taxonomicsubclass rosids. In one aspect, the plant based material is from thetaxonomic order Fabales, such as the family Fabaceae, preferably thesubfamilies Caesalpinioideae or Mimosoideae or Papilionoideae, or morepreferably from the tribes Phaseoleae, Cicereae, Genisteae, Fabeae,Dalbergieae or Phaseoleae. In one aspect, the plant based material isfrom the taxonomic order Brassicales, such as the family Brassicaceae,preferably the tribe Brassiceae, more preferably the family Brassica.

In particular embodiments, the plant based material is soybean, wildsoybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean,lima bean, French bean, Broad bean (fava bean), chickpea, lentil,peanut, Spanish peanut, canola, rapeseed (oilseed rape) or pea or in aprocessed form such as soybean meal, full fat soy bean meal, soy proteinconcentrate (SPC), fermented soybean meal (FSBM) or any combinationthereof. In a preferred embodiment, the plant based material is soybeanor soybean meal.

In a preferred embodiment, the animal feed has been pelleted. The animalfeed may be treated with the enzyme of the invention before thepelleting step or sprayed on after the pelleting step.

Uses

The present invention is also directed to methods for using thepolypeptides having alpha-galactosidase activity, or compositionsthereof, for e.g. animal feed. The present invention is also directed toprocesses for using the polypeptides having alpha-galactosidaseactivity, or compositions thereof, such as e.g. those described below.

Use in Animal Feed

The present invention is also directed to methods for using thealpha-galactosidases of the invention in animal feed.

The term animal includes all animals. Examples of animals arenon-ruminants, and ruminants. Ruminant animals include, for example,animals such as sheep, goats, and cattle, e.g. beef cattle, cows, andyoung calves. In a particular embodiment, the animal is a non-ruminantanimal. Non-ruminant animals include mono-gastric animals, e.g. pigs orswine (including, but not limited to, piglets, growing pigs, and sows);poultry such as turkeys, ducks and chicken (including but not limited tobroiler chicks, layers); horses (including but not limited to hotbloods,coldbloods and warm bloods), young calves; and fish (including but notlimited to salmon, trout, tilapia, catfish and carps; and crustaceans(including but not limited to shrimps and prawns).

In the use according to the invention the alpha-galactosidases can befed to the animal before, after, or simultaneously with the diet. Thelatter is preferred.

In a particular embodiment, the alpha-galactosidase, in the form inwhich it is added to the feed, or when being included in a feedadditive, is well-defined. Well-defined means that thealpha-galactosidase preparation is at least 50% pure as determined bySize-exclusion chromatography (see Example 12 of WO 01/58275). In otherparticular embodiments the alpha-galactosidase preparation is at least60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined bythis method.

A well-defined alpha-galactosidase preparation is advantageous. Forinstance, it is much easier to dose correctly to the feed aalpha-galactosidase that is essentially free from interfering orcontaminating other alpha-galactosidases. The term dose correctly refersin particular to the objective of obtaining consistent and constantresults, and the capability of optimizing dosage based upon the desiredeffect.

For the use in animal feed, however, the alpha-galactosidase need not bethat pure; it may e.g. include other enzymes, in which case it could betermed an alpha-galactosidase preparation.

The alpha-galactosidase preparation can be (a) added directly to thefeed, or (b) it can be used in the production of one or moreintermediate compositions such as feed additives or premixes that issubsequently added to the feed (or used in a treatment process). Thedegree of purity described above refers to the purity of the originalalpha-galactosidase preparation, whether used according to (a) or (b)above.

Preferred Embodiments of the Invention

Preferred embodiments of the invention are described in the set of itemsbelow.

-   1. An animal feed additive comprising one or more GH36 polypeptides    having alpha-galactosidase activity selected from the group    consisting of:    -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        3;    -   (b) a polypeptide encoded by a polynucleotide that hybridizes        under high stringency conditions, or very high stringency        conditions with        -   (i) the mature polypeptide coding sequence of SEQ ID NO: 1,        -   (ii) the cDNA sequence thereof, or        -   (iii) the full-length complementary strand of (i) or (ii);    -   (c) a polypeptide encoded by a polynucleotide having at least        80%, e.g., at least 85%, at least 86%, at least 87%, at least        88%, at least 89%, at least 90%, at least 91%, at least 92%, at        least 93%, at least 94%, at least 95%, at least 96%, at least        97%, at least 98%, at least 99%, or 100% sequence identity to        the mature polypeptide coding sequence of SEQ ID NO: 1;    -   (d) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, wherein the variant has        alpha-galactosidase activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41 42, 43, 44, 45, 46, 47,        48, 49 or 50 positions;    -   (e) a polypeptide comprising the polypeptide of (a), (b), (c)        or (d) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;    -   (f) a polypeptide comprising the polypeptide of (a), (b), (c)        or (d) and a N-terminal and/or C-terminal extension of up to 10        amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;        and    -   (g) a fragment of the polypeptide of (a), (b), (c) or (d) having        alpha-galactosidase activity and having at least 90% of the        length of the mature polypeptide.-   2. The animal feed additive of item 1 wherein the polypeptide has    improved gastric stability compared to control.-   3. The animal feed additive of item 2 wherein control is defined as    SEQ ID NO: 4.-   4. The animal feed additive of any of items 2 to 3 wherein gastric    stability is measured as the half-life at 40° C. pH3 with 0.1 mg/ml    pepsin.-   5. The animal feed additive of item 1 wherein the polypeptide has a    half-life of at least 90 minutes, such as at least 2 hours, 3 hours    or at least 4 hours.-   6. The animal feed additive of item 5 wherein half-life is measured    at 40° C. pH3 with 0.1 mg/ml pepsin.-   7. The animal feed additive of any of items 1 to 6 wherein the    polypeptide has increased alpha-galactosidase activity compared to    control.-   8. The animal feed additive of item 7 wherein control is defined as    SEQ ID NO: 4.-   9. The animal feed additive of any of items 7 to 8 wherein    alpha-galactosidase activity is measured using 4-nitrophenyl    α-D-galactopyranoside as substrate.-   10. The animal feed additive of any of items 7 to 9 wherein    alpha-galactosidase activity is at least 100000 (mOD/min)/(mg/ml),    such as at least 150000, at least 200000, at least 250000, or at    least 300000 (mOD/min)/(mg/ml).-   11. The animal feed additive of any of items 1 to 6, wherein the    polypeptide has at least 40%, such as at least 50%, at least 60%, at    least 70%, at least 75%, at least 80%, at least 85%, at least 90%,    at least 95% or at least 100% of the alpha-galactosidase activity of    the polypeptide of SEQ ID NO: 3.-   12. The animal feed additive of item 11 wherein alpha-galactosidase    activity is measured using 4-nitrophenyl α-D-galactopyranoside as    substrate.-   13. The animal feed additive of any of items 1 to 12 wherein the    polypeptide comprises or consists of amino acids 1 to 721 of SEQ ID    NO: 2 or amino acids 1 to 721 of SEQ ID NO: 3.-   14. The animal feed additive of any of items 1 to 13 further    comprising one or more components selected from the list consisting    of:    -   one or more vitamins;    -   one or more minerals;    -   one or more amino acids;    -   one or more prebiotics;    -   one or more phytogenics;    -   one or more organic acids; and one or more other feed        ingredients.-   15. The animal feed additive of any of items 1 to 14 further    comprising one or more formulating agents.-   16. The animal feed additive of item 15, wherein the one or more    formulating agent is selected from the group consisting of glycerol,    ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol,    sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate,    potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium    carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol,    lactose, starch and cellulose or any combination thereof.-   17. The animal feed additive of any of items 1 to 16 further    comprising one or more additional enzymes.-   18. The animal feed additive of item 17 wherein the one or more    additional enzymes is selected from the group consisting of    acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase,    beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase,    feruloyl esterase, galactanase, alpha-galactosidase,    beta-galactosidase, beta-glucanase, beta-glucosidase,    lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase    (mannanase), phytase, phospholipase A1, phospholipase A2,    phospholipase D, protease, pullulanase, pectinesterase,    triacylglycerol lipase, xylanase, beta-xylosidase or any combination    thereof.-   19 The animal feed additive of any of items 1 to 18 further    comprising one or more microbes.-   20. The animal feed additive of item 19, wherein the one or more    microbes is selected from the group consisting of Bacillus subtilis,    Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus,    Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus    coagulans, Bacillus circulans, Bifidobacterium bifidum,    Bifidobacterium animalis, Bifidobacterium sp., Carnobacterium sp.,    Clostridium butyricum, Clostridium sp., Enterococcus faecium,    Enterococcus sp., Lactobacillus sp., Lactobacillus acidophilus,    Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus    reuteri, Lactobacillus salivarius, Lactococcus lactis, Lactococcus    sp., Leuconostoc sp., Megasphaera elsdenii, Megasphaera sp.,    Pediococsus acidilactici, Pediococcus sp., Propionibacterium    thoenii, Propionibacterium sp. and Streptococcus sp. or any    combination thereof.-   21. An isolated polypeptide having alpha-galactosidase activity,    selected from the group consisting of:    -   (a) a polypeptide having at least 96.5%, e.g., at least 97%, at        least 97.5%, at least 98%, at least 98.5%, at least 99%, at        least 99.5%, or 100% sequence identity to the polypeptide of SEQ        ID NO: 3;    -   (b) a polypeptide encoded by a polynucleotide having at least        96.5%, e.g., at least 97%, at least 97.5%, at least 98%, at        least 98.5%, at least 99%, at least 99.5%, or 100% sequence        identity to the mature polypeptide coding sequence of SEQ ID NO:        1;    -   (c) a variant of the polypeptide selected from the group        consisting of SEQ ID NO: 3, wherein the variant has        alpha-galactosidase activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26 or27 positions;    -   (d) a polypeptide comprising the polypeptide of (a), (b) or (c)        and a N-terminal and/or C-terminal His-tag and/or HQ-tag;    -   (e) a polypeptide comprising the polypeptide of (a), (b) or (c)        and a N-terminal and/or C-terminal extension of up to 10 amino        acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and    -   (f) a fragment of the polypeptide of (a), (b) or (c) having        alpha-galactosidase activity and having at least 90% of the        length of the mature polypeptide.-   22. The polypeptide of item 21, wherein the polypeptide comprises or    consists of amino acids amino acids 1 to 721 of SEQ ID NO: 2 or    amino acids 1 to 721 of SEQ ID NO: 3.-   23. A composition comprising the polypeptide of any of items 21 to    22.-   24. The composition of item 23 further comprising one or more    formulating agents.-   25. The composition of item 24, wherein the one or more formulating    agent is selected from the group consisting of glycerol, ethylene    glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium    chloride, sodium benzoate, potassium sorbate, sodium sulfate,    potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium    carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol,    lactose, starch and cellulose or any combination thereof.-   26. A granule comprising a GH36 polypeptide having    alpha-galactosidase activity, wherein the polypeptide is selected    from the group consisting of:    -   (a) a polypeptide having at least 80%, e.g., at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or 100% sequence identity to the polypeptide of SEQ ID NO:        3;    -   (b) a variant of SEQ ID NO: 3, wherein the variant has        alpha-galactosidase activity and comprises one or more amino        acid substitutions, and/or one or more amino acid deletions,        and/or one or more amino acid insertions or any combination        thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31        32, 33, 34, 35, 36, 37, 38, 39, 40, 41 42, 43, 44, 45, 46, 47,        48, 49 or 50 positions;    -   (c) a polypeptide comprising the polypeptide of (a) or (b) and a        N-terminal and/or C-terminal His-tag and/or HQ-tag;    -   (d) a polypeptide comprising the polypeptide of (a) or (b) and a        N-terminal and/or C-terminal extension of up to 10 amino acids,        e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and    -   (e) a fragment of the polypeptide of (a) or (b) having        alpha-galactosidase activity and having at least 90% of the        length of the mature polypeptide.-   27. A granule comprising the animal feed additive of any of items 1    to 20, one or more polypeptides of any of items 21 to 22 or the    composition of any of items 23 to 25.-   28. The granule of any of items 26 to 27 wherein the granule is    coated.-   29. The granule of item 28 wherein the coating comprises a salt    and/or wax and/or a flour.-   30. A liquid formulation comprising one or more GH36 polypeptides    having alpha-galactosidase activity, wherein the liquid formulation    comprises:    -   (A) 0.001% to 25% w/w of polypeptide having alpha-galactosidase        activity wherein the polypeptide having alpha-galactosidase        activity is selected from the group consisting of:        -   (a) a polypeptide having at least 80%, e.g., at least 85%,            at least 86%, at least 87%, at least 88%, at least 89%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 96%, at least 97%, at            least 98%, at least 99%, or 100% sequence identity to the            polypeptide of SEQ ID NO: 3;        -   (b) a variant of SEQ ID NO: 3, wherein the variant has            alpha-galactosidase activity and comprises one or more amino            acid substitutions, and/or one or more amino acid deletions,            and/or one or more amino acid insertions or any combination            thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,            15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,            30, 31 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 42, 43, 44,            45, 46, 47, 48, 49 or 50 positions;        -   (c) a polypeptide comprising the polypeptide of (a) or (b)            and a N-terminal and/or C-terminal His-tag and/or HQ-tag;        -   (d) a polypeptide comprising the polypeptide of (a) or (b)            and a N-terminal and/or C-terminal extension of up to 10            amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino            acids; and        -   (e) a fragment of the polypeptide of (a) or (b) having            alpha-galactosidase activity and having at least 90% of the            length of the mature polypeptide; and    -   (B) water.-   31. The liquid formulation of item 30, wherein the polypeptide    having alpha-galactosidase activity is dosed between 0.001% to 25%    w/w of liquid formulation, preferably 0.01% to 25% w/w, more    preferably 0.05% to 20% w/w, more preferably 0.2% to 15% w/w, even    more preferably 0.5% to 15% w/w or most preferably 1.0% to 10% w/w    polypeptide.-   32. The liquid formulation of any of items 30 to 31, wherein the    formulation further comprises 20% to 80% w/w of polyol.-   33. The liquid formulation of item 32, wherein the polyol is    selected from the group consisting of glycerol, sorbitol, propylene    glycol (MPG), ethylene glycol, diethylene glycol, triethylene    glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, dipropylene    glycol, polyethylene glycol (PEG) having an average molecular weight    below about 600 and polypropylene glycol (PPG) having an average    molecular weight below about 600 or any combination thereof.-   34. The liquid formulation of any of items 30 to 33, wherein the    formulation further comprises 0.01% to 2.0% w/w preservative.-   35. The liquid formulation of item 34, wherein the preservative is    selected from the group consisting of sodium sorbate, potassium    sorbate, sodium benzoate and potassion benzoate or any combination    thereof.-   36. The liquid formulation of any of items 30 to 35 further    comprising one or more components selected from the list consisting    of:    -   one or more enzymes;    -   one or more microbes;    -   one or more vitamins;    -   one or more minerals;    -   one or more amino acids;    -   one or more phytogenics;    -   one or more prebiotics;    -   one or more organic acids; and one or more other feed        ingredients.-   37. A method of preparing an animal feed comprising applying the    liquid formulation of any of items 30 to 36 onto plant based    material.-   38. The method of item 37, wherein the liquid formulation is applied    via a spray.-   39. The method of any of items 37 to 38, wherein the plant based    material is selected from the group consisting of soybean, wild    soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim    bean, lima bean, French bean, Broad bean (fava bean), chickpea,    lentil, peanut, Spanish peanut, canola, rapeseed (oilseed rape) or    pea or in a processed form such as soybean meal, full fat soy bean    meal, soy protein concentrate (SPC), fermented soybean meal (FSBM)    or any combination thereof.-   40. The method of any of items 37 to 39, wherein the plant based    material is in pelleted form.-   41. A pelleted animal feed prepared using the method of any of items    37 to 40.-   42. An animal feed comprising plant based material and the animal    feed additive of any of items 1 to 20, one or more polypeptides of    any of items 21 to 22, the composition of any of items 23 to 25, the    granule of any of items 26 to 29 or the liquid formulation of any of    items 30 to 36.-   43. The animal feed of item 42, wherein the plant based material is    from the taxonomic subclass rosids.-   44. The animal feed of item 42, wherein the plant based material is    from the family Fabaceae, preferably the sub-family Papilionoideae,    more preferably from the tribe Phaseoleae, Cicereae, Genisteae,    Fabeae, Dalbergieae or Phaseoleae or any combination thereof-   45. The animal feed of item 42, wherein the plant based material is    soybean, wild soybean, beans, lupin, tepary bean, scarlet runner    bean, slimjim bean, lima bean, French bean, Broad bean (fava bean),    chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (oilseed    rape) or pea or in a processed form such as soybean meal, full fat    soy bean meal, soy protein concentrate (SPC), fermented soybean meal    (FSBM) or any combination thereof.-   46. A pelleted animal feed comprising plant based material and the    animal feed additive of any of items 1 to 20, one or more    polypeptides of any of items 21 to 22, the composition of any of    items 23 to 25, the granule of any of items 26 to 29 or the liquid    formulation of any of items 30 to 36.-   47. The pelleted animal feed of item 46, wherein the plant based    material is from the subclass rosids, preferably the family    Fabaceae, more preferably the sub-family Papilionoideae or more    preferably is from the tribe Phaseoleae, Cicereae, Genisteae,    Fabeae, Dalbergieae or Phaseoleae or any combination thereof.-   48. The pelleted animal feed of item 46, wherein the plant based    material is soybean, wild soybean, beans, lupin, tepary bean,    scarlet runner bean, slimjim bean, lima bean, French bean, Broad    bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola,    rapeseed (oilseed rape) or pea or in a processed form such as    soybean meal, full fat soy bean meal, soy protein concentrate (SPC),    fermented soybean meal (FSBM) or any combination thereof.-   49. A method of releasing galactose from plant based material,    comprising treating the plant based material with the animal feed    additive of any of items 1 to 20, one or more polypeptides of any of    items 21 to 22, the composition of any of items 23 to 25, the    granule of any of items 26 to 29 or the liquid formulation of any of    items 30 to 36.-   50. A method of improving one or more performance parameters of an    animal comprising administering to one or more animals the animal    feed additive of any of items 1 to 20, one or more polypeptides of    any of items 21 to 22, the composition of any of items 23 to 25, the    granule of any of items 26 to 29, the liquid formulation of any of    items 30 to 36, the animal feed of any of items 42 to 45 or the    pelleted animal feed of any of items 41 or 46 to 48.-   51. The method of item 50, wherein the performance parameter is    selected from the list consisting of body weight gain (BWG),    European Production Efficiency Factor (EPEF) and Feed Conversion    Ratio (FCR) or any combination thereof.-   52. A method for improving the nutritional value of an animal feed    comprising plant based material, comprising mixing the plant based    material with the animal feed additive of any of items 1 to 20, one    or more polypeptides of any of items 21 to 22, the composition of    any of items 23 to 25, the granule of any of items 26 to 29 or the    liquid formulation of any of items 30 to 36.-   53. A method of preparing an animal feed, comprising mixing the    animal feed additive of any of items 1 to 20, one or more    polypeptides of any of items 21 to 22, the composition of any of    items 23 to 25, the granule of any of items 26 to 29 or the liquid    formulation of any of items 30 to 36 with plant based material.-   54. The methods of any of items 49 to 53, wherein the plant based    material is from the taxonomic subclass rosids.-   55. The methods of any of items 49 to 53, wherein the plant based    material is from the family Fabaceae, preferably the sub-family    Papilionoideae, more preferably from the tribe Phaseoleae, Cicereae,    Genisteae, Fabeae, Dalbergieae or Phaseoleae or any combination    thereof.-   56. The methods of any of items 49 to 53, wherein the plant based    material is soybean, wild soybean, beans, lupin, tepary bean,    scarlet runner bean, slimjim bean, lima bean, French bean, Broad    bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola,    rapeseed (oilseed rape) or pea or in a processed form such as    soybean meal, full fat soy bean meal, soy protein concentrate (SPC),    fermented soybean meal (FSBM) or any combination thereof.-   57. Use of the animal feed additive of any of items 1 to 20, one or    more polypeptides of any of items 21 to 22, the composition of any    of items 23 to 25, the granule of any of items 26 to 29, the liquid    formulation of any of items 30 to 36, the animal feed of any of    items 42 to 45 or the pelleted animal feed of any of items 41 or 46    to 48:    -   in animal feed;    -   in animal feed additives;    -   in the preparation of a composition for use in animal feed;    -   for improving the nutritional value of an animal feed;    -   for increasing digestibility of the animal feed;    -   for improving one or more performance parameters in an animal;        and/or for releasing galactose from plant based material.-   58 A polynucleotide encoding the polypeptide of any of items 21 to    22.-   59. A nucleic acid construct or expression vector comprising the    polynucleotide of item 58 operably linked to one or more control    sequences that direct the production of the polypeptide in an    expression host.-   60. A recombinant host cell comprising the polynucleotide of item 58    operably linked to one or more control sequences that direct the    production of the polypeptide.-   61. A method of producing the polypeptide of any of items 21 to 22,    comprising:    -   (a) cultivating a cell, which in its wild-type form produces the        polypeptide, under conditions conductive for production of the        polypeptide; and    -   (b) recovering the polypeptide.-   62. A method of producing the polypeptide of any of items 21 to 22,    comprising:    -   (a) cultivating the recombinant host cell of item 60 under        conditions conducive for production of the polypeptide; and    -   (b) recovering the polypeptide.-   63. A transgenic plant, plant part or plant cell transformed with a    polynucleotide encoding the polypeptide of any of items 21 to 22.-   64. A whole broth formulation or cell culture composition comprising    a polypeptide of any of items 21 to 22.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Strains

The alpha-galactosidases were derived from bacterial strains isolatedfrom environmental samples by standard microbiological isolationtechniques. Strains were identified and taxonomy was assigned based onDNA sequencing of the 16S ribosomal genes (Table 2).

TABLE 2 Isolation of fungal strain SEQ ID SEQ ID NO of NO of poly-Strain Source Country Year gene peptide Penicillium Soil Nicaragua 19491 2 pseudopulvillorum

Chromosomal DNA isolated from pure cultures of the individual strainswith the DNeasy Blood & Tissue Kit from Qiagen (Hilden, Germany) wassubjected to full genome sequencing using Illumina technology. Genomesequencing, the subsequent assembly of reads and the gene discovery(i.e. annotation of gene functions) is known to the person skilled inthe art and the service can be purchased commercially.

The genome sequence was analyzed for alpha-galactosidases from the CAZYdatabase family GH36 (Lombard et al. The Carbohydrate-active enzymesdatabase CAZy. Nucleic Acids Res 2013, 42:D490-D495.) This analysisidentified a gene encoding a putative secreted alpha-galactosidase withthe nucleotide sequence given in SEQ ID NO: 1.

Alpha-Galactosidase Assay

Alpha-galactosidase activity can be determined using 4-nitrophenylα-D-galactopyranoside (product code O-PNPBGAL, available from MegazymeInternational, Bray, Co. Wicklow, Ireland) as follows.

The enzyme was diluted using 100 mM MES (Sigma) buffer pH 7.0±0.05 in2-fold dilutions and then the 4-nitrophenyl α-D-galactopyranoside (1mg/ml in 100 mM MES buffer pH 7.0±0.05, prepared immediately before use)was added in the solution containing the enzyme. The respectivegalactosidase activity was followed directly in the buffer by measuringthe absorbance of released pNP (para-nitro-phenol) at 405 nm for 5minutes as function of time at room temperature (typically 23° C.). Aconcentration of 1 mg/mL of enzyme is a good starting point; it willhowever depend from enzyme to enzyme and their specific activity.

The activity is calculated as the slope of a plot of absorbance versustime (units: mOD/min) using the 1-5 minute time window and the 0-2absorbance window. The activity can then be converted to specificactivity by dividing the activity for the concentration of the enzyme(units: (mOD/min)/(mg/ml)).

Galactose Assay

Introduction

The concentration of galactose monosaccharides in a solution wasmeasured spectrophotometrically after enzymatic hydrolysis of agalactose-rich substrate; soybean meal.

Summarizing, the enzyme(s) were incubated in a 10 w/v % slurry ofsoybean meal at pH 6.5±0.05 for 2 hours at 40±2° C. The supernatant wasthen analyzed in an assay based on the Raffinose/Galactose kit fromMegazyme (product name K-RAFGA). First α-D-galactose in the supernatantwas converted to β-D-galactose with the enzyme galactose mutarotase.Then 1-D-galactose was oxidised by NAD+ to D-galactonic acid in thepresence of β-galactose dehydrogenase. The amount of NADH formed in thisreaction was stoichiometric with the amount of D-Galactose in thesupernatant. NADH concentration was then measured by the increase inabsorbance at 340 nm.

Soybean Meal Slurry

A 10 w/v % slurry of soybean meal was prepared from soybean meal milledto a 0.5 mm particle size and 0.1 M citric acid-phosphate buffer, pH6.5±0.05.

0.1 M citric acid-phosphate buffer, pH 6.5±0.05 was heated to atemperature of approximately 40° C. while stirring. The preheated bufferwas then transferred to the soybean meal. The resulting slurry wasstirred while being heated (temperature was not monitored at thispoint—heating was only applied to ensure that temperature would notdecrease too much while the slurry stirred). The slurry was thentransferred with a pre-wetted wide-bore pipette to the vessel in whichit should be incubated. The slurry was pipetted from an approximatelycentral point in the mix. The time elapsed from the mixing of the slurryuntil transfer to the last incubation vessel was, at most, 15 minutes.Stirring speed was adjusted in such a way that particles were evenlydistributed in the slurry.

Dilution of Enzymes

The enzymes were diluted to their desired concentrations in ultrapurewater. The concentration to which the enzymes were diluted to was basedon the prior concentration of the enzyme in mg enzyme protein per mL (mgEP/mL) and the mass (kg) of dry matter (soybean meal) in each incubationvessel.

${V_{enzyme}({mL})} = \frac{c_{enzyme}\left( {{mg}\frac{EP}{mL}} \right)}{m_{SBM}({kg})}$D-(+)-galactose standards

A standard curve was prepared from D-(+)-galactose and ultrapure water.A D-(+)-galactose stock was prepared by dissolving D-(+)-galactose inultrapure water to a final concentration of 250 mg galactose per mL. Thestock solution was diluted in a two-fold dilution row to obtain sixstandards with concentrations of 250, 125, 62.5, 31.25, 15.625 and 7.813mg galactose per mL.

Incubation of α-Galactosidases on Soybean Meal

The incubation vessels with the 10 w/v % slurry of soybean meal wereheated to a stable temperature of 40±2° C. while stirring. When a stabletemperature had been achieved, the six D-(+)-galactose standards wereadded to the incubation vessels to in-vessel concentrations of 5, 2.5,1.25, 0.625, 0.313 and 0.157 mg galactose per mL incubation volume. Eachstandard was incubated in duplicates.

The diluted enzymes were then added to their respective incubationvessels in the volumes required to reach their desired concentrations(in mg EP/kg soybean meal). Each enzyme treatment was incubated intriplicates.

Additionally, two times three incubation vessels were included withoutstandards or enzyme treatments as blank treatments to obtain thebaseline galactose concentration in the soybean meal slurry.

The incubation vessels were incubated at 40±2° C., while stirring for 2hours. After incubation the vessels were centrifuged at 1500 g at 5° C.for 15 minutes.

Determination of Galactose Concentration

The supernatants in the now centrifuged incubation vessels were thenanalyzed in an assay based on the Raffinose/Galactose kit from Megazyme(product name K-RAFGA). Three reagents from the K-RAFGA kit was used inthe assay: Assay Buffer (supplied and ready in Bottle 1 in the kit),1-NAD reagent (supplied in Bottle 2 in the kit, prepared as described inthe kit prior to use) and GalDH+GalM solution (supplied in Bottle 3 inthe kit, diluted 1:1 in ultrapure water prior to use). All stepsdescribed in the following were carried out using an Eppendorf 5075automated pipetting system.

First the supernatants from the centrifuged incubation vessels werediluted 10 times in 0.1 M citric acid-phosphate buffer, pH 6.5±0.05 (1part supernatant plus 9 parts 0.1 M citric acid-phosphate buffer, pH6.5±0.05).

69 μL of each diluted supernatant was then transferred to a new vesseland 34 μL of ultrapure water was added to the diluted supernatants(which will be referred to as assay samples from here on out). Then 69μL Assay Buffer was added to the assay samples followed by dilution in687 μL ultrapure water. 34 μL 1-NAD reagent was added to the assaysamples, followed by addition of 14 μL GalDH+GalM solution and vigorousmixing.

262 μL of each assay sample was then transferred to a 96 well microtiter plate. Absorbance in each well of the 96 well micro titer platewas measured at 340 nm at 40±2° C. for a duration of 20 minutes or untilabsorbance in each well had reached a stable level. When a stableabsorbance had been reached this stable absorbance was used in latercalculations.

Calculation of Galactose Concentration

Absorbance of the assay samples from the galactose standards in theincubation vessels were used as a standard curve (6 standards, 5, 2.5,1.25, 0.625, 0.313 and 0.157 mg galactose per mL incubation volume, n=2per standard). An equation for the galactose standard curve wascalculated in excel, where y is OD340 and x is galactose concentrationin mg galactose per mL incubation volume:

${OD}_{340} = {{a*{c_{gal}\left( \frac{mg}{mL} \right)}} + b}$

Galactose concentration in mg galactose per mL incubation volume foreach sample was then given by:

${c_{gal}\left( \frac{mg}{mL} \right)} = \frac{{OD}_{340} - b}{a}$

Galactose concentrations were then calculated on a dry-matter basis (ggalactose per kg soybean meal) and are reported in the examples below:

${c_{gal}\left( {\frac{g}{kg}{SBM}} \right)} = \frac{{c_{gal}\left( \frac{mg}{mL} \right)}*{V_{sample}({mL})}}{m_{SBM}(g)}$

Example 1: Cloning of GH36 Alpha-Galactosidase (SEQ ID NO: 1)

The alpha-galactosidase with nucleotide sequence SEQ ID NO: 1 and thepeptide translation of the protein shown in SEQ ID NO: 2 was PCRamplified from genomic DNA isolated from Penicillium pseudopulvillorumand cloned into the expression vector pDAu222 as described in WO2013024021 using BamHI and XhoI restriction sites.

The sequence of the alpha-galactosidase encoding gene cloned in theexpression vector was confirmed and the expression construct wastransformed into the Aspergillus oryzae strain MT3568 (WO 11/057140) toproduce the secreted mature peptide with protein sequence SEQ ID NO: 12.Transformants were selected on acetamide during regeneration fromprotoplasts and subsequently re-isolated under selection (Christensen etal., 1988, Biotechnology 6, 1419-1422 and WO 04/032648).

For production of the recombinant alpha-galactosidase, a singleAspergillus transformant was cultured in two 500 ml baffled flasks eachcontaining 150 ml of DAP-4C-1 medium (WO 12/103350). The cultures wereshaken on a rotary table at 100 RPM at 30° C. for 4 days. The culturebroth subsequently was separated from cellular material by passagethrough a 0.22 um filter.

Example 2: Purification of GH36 Alpha-Galactosidases (SEQ ID NO: 3)

Filtrated broth was adjusted to pH7.0 and filtrated on 0.22 μm PESfilter (Nalge Nunc International, Nalgene labware cat #595-4520).Following, the filtrate was added 1.8M ammonium sulphate. The filtratewas loaded onto a Phenyl Sepharose™ 6 Fast Flow column (high sub) (GEHealthcare, Piscataway, N.J., USA) equilibrated with 1.2M ammoniumsulphate, 25 mM HEPES pH7.0. After wash with 1.0M ammonium sulphate, thebound proteins were batch eluted with 25 mM HEPES pH 7.0. Fractions werecollected and analyzed by SDS-PAGE. The fractions were pooled andapplied to a Sephadex™ G-25 (medium) (GE Healthcare, Piscataway, N.J.,USA) column equilibrated in 25 mM HEPES pH 7.5. The fractions wereapplied to a SOURCE™ 15Q (GE Healthcare, Piscataway, N.J., USA) columnequilibrated in 25 mM HEPES pH 7 and bound proteins were eluted with 25mM HEPES pH 7, 1 M sodium chloride over ca. 20CV. Fractions werecollected and analyzed by SDS-PAGE.

Example 3: Specific Activity of the GH36 Alpha-Galactosidase of SEQ IDNO: 3

The specific activity of the GH36 alpha-galactosidase of the invention(SEQ ID NO: 3) was measured using the Alpha-Galactosidase Assay asdisclosed herein and compared to the activity of the prior art GH36alpha-galactosidase from Aspergillus niger (SEQ ID NO: 4). The resultsare presented in table 3.

TABLE 3 Specific activity of the GH36 alpha- galactosidases (SEQ ID NO:3 and 4) GH36 alpha- Specific Activity on pNP substrate galactosidase(mOD/min)/(mg/ml) SEQ ID NO: 3 360000 SEQ ID NO: 4 85000

The GH36 alpha-galactosidase of the invention (SEQ ID NO: 3) issignificantly more active in releasing pNP from 4-nitrophenylα-D-galactopyranoside than the prior art alpha-galactosidase of SEQ IDNO: 4.

Example 4: Gastric Stability of the GH36 Alpha-Galactosidase of SEQ IDNO: 3

Reagents

Pepsin from porcine gastric mucosa (Sigma P7000) diluted in 50 mMglycine buffer pH 3.00±0.05

Stressing buffer 1: 50 mM glycine pH 3.00±0.05

Stressing buffer 2: 50 mM glycine pH 3.00±0.05+0.1 mg/mL pepsin

Stressing buffer 3: 100 mM MES pH 7.00±0.05

Recovery buffer: 100 mM MES pH 7.00±0.05

Substrate working solution: 1 mg/mL α-pNP diluted in 100 mM MES pH7.00±0.05. This working solution is made immediately before use.

Procedure

160 μL of stressing buffer 1, stressing buffer 2 or stressing buffer 3was added to the wells of a 96 well PCR plate. The PCR plate wasincubated at 40° C. for 10 minutes, then 40 μL of thealpha-galactosidase sample to be analyzed was added to the wellscontaining the stressing buffer. Note that the enzyme was diluted sothat the activity in stressing buffer 3 was −250 mOD/min. After 2, 5,10, 15, 20, 25 and 30 minutes incubation at 40° C., 20 μL of thestressed enzymes was added to 80 μL of recovery buffer in the wells of amicro titer plate kept at room temperature. 100 μL of substrate workingsolution was then added to the wells of the micro titer plate. Thesolution was mixed for 10 seconds at room temperature and absorption at405 nm was measured every 30 seconds over 5 minutes.

The activity of the α-galactosidase (V0) was calculated as the slope ofthe time-dependent absorption curve in the 0.0-2.0 OD absorbance window.In order to evaluate the half-life of the enzyme, the activity (V0) wasplotted against the incubation time and the data was fitted to anexponential function of the type: V0(t)=A e−λt, where V0(t) is theactivity V0 at time t, A=V0(0) is the V0 at time=0 and A is theexponential decay constant (also known as decay rate). The half-life isthen evaluated as T=(In(2))A.

The results are presented in table 4.

TABLE 4 Specific activity of the GH36 alpha- galactosidases (SEQ ID NO:3 and 4) T½ (min) T½ (min) GH36 alpha- 40 C. pH 3 no 40 C. pH 3 with 0.1mg/ml galactosidase pepsin pepsin SEQ ID NO: 3 Stable Stable SEQ ID NO:4 Stable 77 ± 1

The results show that whilst the prior art alpha-galactanase (SEQ ID NO:4) is stable at pH3 without any pepsin being present, it decomposesrelatively quickly in the presence of 0.1 mg/ml pepsin. In comparison,the alpha-galactanase of the invention (SEQ ID NO: 3) is completelystable both with and without pepsin present.

Example 5: Animal Feed and Animal Feed Additives Comprising anAlpha-Galactosidase

Animal Feed Additive

A formulation of an alpha-galactosidase of the invention (e.g. SEQ IDNO: 3) containing 0.01 g to 10 g enzyme protein is added to thefollowing premix (per kilo of premix):

5000000 IE Vitamin A 1000000 IE Vitamin D3 13333 mg Vitamin E 1000 mgVitamin K3 750 mg Vitamin B1 2500 mg Vitamin B2 1500 mg Vitamin B6 7666mcg Vitamin B12 12333 mg Niacin 33333 mcg Biotin 300 mg Folic Acid 3000mg Ca-D-Panthothenate 1666 mg Cu 16666 mg Fe 16666 mg Zn 23333 mg Mn 133mg Co 66 mg I 66 mg Se 5.8 % Calcium 25 % SodiumAnimal Feed

This is an example of an animal feed (broiler feed) comprising theanimal feed additive as described above:

62.55% Maize

33.8% Soybean meal (50% crude protein)

1.0% Soybean oil

0.2% DL-Methionine

0.22% DCP (dicalcium phosphate)

0.76% CaCO₃ (calcium carbonate)

0.32% Sand

0.15% NaCl (sodium chloride)

1% of the above Premix

The ingredients are mixed, and the feed is pelleted at the desiredtemperature, e.g. 60, 65, 75, 80, 85, 90 or even 95° C.

Liquid Formulation

A liquid formulation of an alpha-galactosidase of the invention (e.g.SEQ ID NO: 3) comprises 0.1% to 10 w/w enzyme protein, 40-60% glycerol,0.1 to 0.5% sodium benzoate and water. The liquid formulation is sprayedonto the pelleted animal feed described above (in this case the animalfeed additive would not include the alpha-galactosidase of the inventionpresent).

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. An animal feed comprising a plant-based materialand a Glycoside Hydrolase 36 (GH36) polypeptide havingalpha-galactosidase activity, wherein the GH36 polypeptide has at least98% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and wherein the GH36 polypeptide has improved gastric stabilityand/or increased alpha-galactosidase activity using 4-nitrophenylα-D-galactopyranoside as substrate compared to control and is in theform of a granule.
 2. The animal feed of claim 1, wherein theplant-based material is selected from the group consisting of soybean,wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjimbean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil,peanut, Spanish peanut, canola, rapeseed (oilseed rape), pea, soybeanmeal, full fat soy bean meal, soy protein concentrate (SPC), fermentedsoybean meal (FSBM) and any combination thereof.
 3. The animal feed ofclaim 1, wherein the plant-based material is soybean or soybean meal. 4.The animal feed of claim 1, wherein the GH36 polypeptide has at least99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3.
 5. The animal feed of claim 1, wherein the GH36 polypeptide comprisesthe amino acid sequence set forth in SEQ ID NO:
 3. 6. The animal feed ofclaim 1, wherein the GH36 polypeptide consists of the amino acidsequence set forth in SEQ ID NO:
 3. 7. The animal feed of claim 1,wherein the GH36 polypeptide comprises an N-terminal and/or C-terminalHis-tag and/or HQ-tag.
 8. The animal feed of claim 1, further comprisingone or more components selected from the group consisting of: one ormore vitamins; one or more minerals; one or more amino acids; one ormore prebiotics; one or more phytogenics; one or more organic acids; andone or more other feed ingredients.
 9. The animal feed of claim 1,further comprising one or more additional enzymes.
 10. The animal feedof claim 9, wherein the one or more additional enzymes are selected fromthe group consisting of acetylxylan esterase, acylglycerol lipase,alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolase,cellulase, feruloyl esterase, galactanase, alpha-galactosidase,beta-galactosidase, beta-glucanase, beta-glucosidase, lysophospholipase,lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase,phospholipase A1, phospholipase A2, phospholipase D, protease,pullulanase, pectinesterase, triacylglycerol lipase, xylanase,beta-xylosidase and any combination thereof.
 11. A method of improvingone or more performance parameters of an animal, comprisingadministering to one or more animals the animal feed of claim 1, whereinthe one or more performance parameters are increase in body weight gain,improved feed conversion ratio, or increased feed efficiency.
 12. Aliquid formulation comprising water, a polyol, and a Glycoside Hydrolase36 (GH36) polypeptide having alpha-galactosidase activity, wherein theGH36 polypeptide has at least 98% sequence identity to the amino acidsequence set forth in SEQ ID NO: 3, and wherein the GH36 polypeptide hasimproved gastric stability and/or increased alpha-galactosidase activityusing 4-nitrophenyl α-D-galactopyranoside as substrate compared tocontrol.
 13. The liquid formulation of claim 12, wherein the polyol isselected from the group consisting of glycerol, sorbitol, propyleneglycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1, 3-propylene glycol, dipropylene glycol,polyethylene glycol (PEG) having an average molecular weight below about600 and polypropylene glycol (PPG) having an average molecular weightbelow about 600 and any combination thereof.
 14. The liquid formulationof claim 12, further comprising 0.01% to 2.0% w/w preservative, whereinthe preservative is selected from the group consisting of sodiumsorbate, potassium sorbate, sodium benzoate and potassium benzoate andany combination thereof.
 15. The liquid formulation of claim 12, furthercomprising one or more components selected from the group consisting of:one or more enzymes; one or more microbes; one or more vitamins; one ormore minerals; one or more amino acids; one or more phytogenics; one ormore prebiotics; one or more organic acids; and one or more other feedingredients.
 16. A method of preparing an animal feed, comprisingapplying the liquid formulation of claim 12 to a plant-based material.17. The method of claim 16, wherein the plant based material is selectedfrom the group consisting of soybean, wild soybean, beans, lupin, teparybean, scarlet runner bean, slimjim bean, lima bean, French bean, Broadbean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola,rapeseed (oilseed rape), pea, soybean meal, full fat soy bean meal, soyprotein concentrate (SPC), fermented soybean meal (FSBM) and anycombination thereof.